Antibodies against clostridium difficile toxins and uses thereof

ABSTRACT

Antibodies that specifically bind to toxins of C. difficile, antigen binding portions thereof, and methods of making and using the antibodies and antigen binding portions thereof are provided herein.

RELATED INFORMATION

The application is a continuation of U.S. patent application Ser. No.15/924,703, filed on Mar. 19, 2018, which is a continuation of U.S.patent application Ser. No. 14/926,706, filed on Oct. 29, 2015, which isa continuation of U.S. patent application Ser. No. 14/080,598, filed onNov. 14, 2013, now U.S. Pat. No. 9,217,029, which is a divisionalapplication of U.S. patent application Ser. No. 13/490,757, filed onJun. 7, 2012, now U.S. Pat. No. 8,609,111, which is a continuation ofU.S. patent application Ser. No. 12/533,501, filed on Jul. 31, 2009, nowU.S. Pat. No. 8,236,311, which is a divisional application of U.S.patent application Ser. No. 11/051,453, filed on Feb. 4, 2005, now U.S.Pat. No. 7,625,559, which claims priority to U.S. provisional patentapplication No. 60/542,357, filed on Feb. 6, 2004, and U.S. provisionalpatent application No. 60/613,854, filed on Sep. 28, 2004, the entirecontents both of which are hereby incorporated by reference.

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 16, 2020, isnamed MJI-001DV1CNDVCN3_Sequence_Listing.txt and is 99,987 bytes insize.

BACKGROUND OF THE INVENTION

Clostridium difficile (C. difficile) is a gram-positive bacterium thatcauses gastrointestinal disease in humans. C. difficile is the mostcommon cause of infectious diarrhea in hospital patients, and is one ofthe most common nosocomial infections overall (Kelly et al., New Eng. J.Med., 330:257-62, 1994). In fact, disease associated with this pathogenmay afflict as many as three million hospitalized patients per year inthe United States (McFarland et al., New Eng. J. Med., 320:204-10, 1989;Johnson et al., Lancet, 336:97-100, 1990).

Treatment with antibiotics such as ampicillin, amoxicillin,cephalosporins, and clindamycin that disrupt normal intestinal flora canallow colonization of the gut with C. difficile and lead to C. difficiledisease (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998). The onsetof C. difficile disease typically occurs four to nine days afterantibiotic treatment begins, but can also occur after discontinuation ofantibiotic therapy. C. difficile can produce symptoms ranging from mildto severe diarrhea and colitis, including pseudomembranous colitis(PMC), a severe form of colitis characterized by abdominal pain, waterydiarrhea, and systemic illness (e.g., fever, nausea). Relapsing diseasecan occur in up to 20% of patients treated for a first episode ofdisease, and those who relapse are at a greater risk for additionalrelapses (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).

C. difficile disease is believed to be caused by the actions of twoexotoxins, toxin A and toxin B, on gut epithelium. Both toxins are highmolecular weight proteins (280-300 kDa) that catalyze covalentmodification of Rho proteins, small GTP-binding proteins involved inactin polymerization, in host cells. Modification of Rho proteins by thetoxins inactivates them, leading to depolymerization of actin filamentsand cell death. Both toxins are lethal to mice when injectedparenterally (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).

C. difficile disease can be diagnosed by assays that detect the presenceor activity of toxin A or toxin B in stool samples, e.g., enzymeimmunoassays. Cytotoxin assays can be used to detect toxin activity. Toperform a cytotoxin assay, stool is filtered to remove bacteria, and thecytopathic effects of toxins on cultured cells are determined (Merz etal., J. Clin. Microbiol., 32:1142-47, 1994).

C. difficile treatment is complicated by the fact that antibioticstrigger C. difficile associated disease. Nevertheless, antibiotics arethe primary treatment option at present. Antibiotics least likely tocause C. difficile associated disease such as vancomycin andmetronidazole are frequently used. Vancomycin resistance evolving inother microorganisms is a cause for concern in using this antibiotic fortreatment, as it is the only effective treatment for infection withother microorganisms (Gerding, Curr. Top. Microbiol. Immunol.,250:127-39, 2000). Probiotic approaches, in which a subject isadministered non-pathogenic microorganisms that presumably compete forniches with the pathogenic bacteria, are also used. For example,treatment with a combination of vancomycin and Saccharomyces boulardiihas been reported (McFarland et al., JAMA., 271(24):1913-8, 1994.Erratum in: JAMA, 272(7):518, 1994).

Vaccines have been developed that protect animals from lethal challengein infectious models of disease (Torres et al., Infect. Immun.63(12):4619-27, 1995). In addition, polyclonal antibodies have beenshown to protect hamsters from disease when administered by injection orfeeding (Giannasca et al., Infect. Immun. 67(2):527-38, 1999; Kink andWilliams, Infect. Immun., 66(5):2018-25, 1998). Murine monoclonalantibodies have been isolated that bind to C. difficile toxins andneutralize their activities in vivo and in vitro (Corthier et al.,Infect. Immun., 59(3):1192-5, 1991). There are some reports that humanpolyclonal antibodies containing toxin neutralizing antibodies canprevent C. difficile relapse (Salcedo et al., Gut., 41(3):366-70, 1997).Antibody response against toxin A has been correlated with diseaseoutcome, indicating the efficacy of humoral responses in controllinginfection. Individuals with robust toxin A ELISA responses had lesssevere disease compared to individuals with low toxin A antibody levels(Kyne et al., Lancet, 357(9251):189-93, 2001).

The individual role of toxin A and toxin B in disease pathogenesis, andthe role of anti-toxin antibodies in protection from C. difficiledisease are controversial and may depend on the host. In humans, theanti-toxin A antibody response has been correlated to disease outcome,suggesting a requirement for anti-toxin A response for protection. Thisobservation is in contrast with reports of disease-causing C. difficileorganisms that express only toxin B, implying that toxin B cancontribute to disease in humans. These toxin A-negative strains can alsocause disease in hamsters (Sambol et al., J. Infect. Dis.,183(12):1760-6, 2001).

SUMMARY OF THE INVENTION

This invention is based, in part, on the discovery that administrationof antibodies against C. difficile toxin A to a subject can protect thesubject from relapse of C. difficile-mediated disease in vivo.Administration of antibodies to one or both of toxin A and toxin B canprevent primary C. difficile-mediated disease. High affinity antibodiesagainst C. difficile toxins can be produced, e.g., in mice, such astransgenic mice expressing human immunoglobulin gene segments. Theseantibodies can neutralize toxin cytotoxicity in vitro, and neutralizetoxin enterotoxicity in vivo. Antibodies that recognize toxin A and/ortoxin B can inhibit and protect from disease in vivo.

In one aspect, the invention features isolated human monoclonalantibodies or antigen binding portions thereof that specifically bind toan exotoxin of Clostridium difficile (C. difficile). In certainembodiments, the antibodies or antigen binding portions thereofspecifically bind to C. difficile toxin A (toxin A). In otherembodiments, the antibody or antigen binding portions thereofspecifically bind to C. difficile toxin B (toxin B). In otherembodiments, the antibodies or antigen binding portions thereofspecifically bind to both toxin A and toxin B.

In certain embodiments, the antibodies or antigen binding portionsthereof neutralize toxin A in vitro, inhibit binding of toxin A tomammalian cells, and/or inhibit C. difficile-mediated disease in vivo.

In various embodiments, the antibodies or antigen binding portionsthereof have one or more of the following characteristics: whenadministered to a mouse, they protect the mouse against administrationof a C. difficile toxin in an amount that would be fatal to a controlmouse not administered the antibody; protect from or inhibit C.difficile-mediated colitis, antibiotic-associated colitis, orpseudomembranous colitis (PMC) in a subject; protect from or inhibitdiarrhea in a subject; and/or inhibit relapse of C. difficile-mediateddisease.

The antibodies or antigen binding portions thereof can specifically bindto an epitope within the N-terminal half of toxin A, e.g., an epitopebetween amino acids 1-1256 of toxin A. In other embodiments, theantibodies or antigen binding portions thereof specifically bind to anepitope within the C-terminal receptor binding domain of toxin A, e.g.,an epitope between amino acids 1852-2710 of toxin A, or an epitopebetween amino acids 659-1852, e.g., an epitope within amino acidresidues 900-1852, 900-1200, or 920-1033 of toxin A. In otherembodiments, the antibodies or antigen binding portions thereofspecifically bind an epitope within amino acids 1-600, 400-600, or415-540 of toxin A. Other particular antibodies or antigen bindingportions thereof, can specifically bind to an epitope within amino acidresidues 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1800-1900, 1900-200, 2100-2200 or 2200-2300,2300-2400, 2400-2500, 2500-2600, 2600-2710 of toxin A, or any interval,portion or range thereof.

In certain embodiments, the antibodies or antigen binding portionsthereof specifically bind to toxin A with a K_(D) of less than about20×10⁻⁶ M. In a particular embodiment, the antibody, or antigen bindingportion thereof, specifically binds to toxin A with a K_(D) of less thanabout 10×10⁻⁷ M, less than about 10×10⁻⁸ M, less than about 10×10⁻⁹ M,or less than about 10×10⁻¹⁰ M. In other particular embodiments, theantibody, or antigen binding portion thereof, specifically binds totoxin A with a K_(D) of less than about 50×10⁻¹⁰ M, less than about20×10⁻¹⁰ M, less than about 15×10⁻¹⁰ M, less than about 8×10⁻¹⁰ M, orless than about 5×10⁻¹⁰ M.

In various other embodiments, the antibodies or antigen binding portionsthereof include a variable heavy chain region including an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to avariable heavy chain region amino acid sequence of the antibody producedby clone 3D8 (SEQ ID NO:1), 1B11 (SEQ ID NO:2), or 3H2 (SEQ ID NO:3).

In certain embodiments, the antibodies or antigen binding portionsthereof include a variable light chain region comprising an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to avariable light chain region amino acid sequence of the antibody producedby clone 3D8 (SEQ ID NO:4), 1B11 (SEQ ID NO:5), or 3H2 (SEQ ID NO:6).

In certain embodiments, the antibodies or antigen binding portionsthereof each include both a variable heavy chain region including anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or moreidentical to a variable heavy chain region amino acid sequence of theantibody produced by clone 3D8 (SEQ ID NO:1), 1B11 (SEQ ID NO:2), or 3H2(SEQ ID NO:3), and a variable light chain region including an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to avariable light chain amino acid sequence of clone 3D8 (SEQ ID NO:4),1B11 (SEQ ID NO:5), or 3H2 (SEQ ID NO:6).

In various embodiments, the antibodies or antigen binding portionsthereof specifically bind to an epitope that overlaps with an epitopebound by an antibody produced by clone 3D8, 1B11, or 3H2 and/or competefor binding to toxin A with an antibody produced by clone 3D8, 1B11, or3H2.

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include one or more complementarity determiningregions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of the antibody produced by clone 3D8 (SEQ IDNOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15) (also shownin Table 1).

A variable light chain region of the antibodies or antigen bindingportions thereof can include one or more CDRs that are at least 80%,85%, 90%, 95%, or 99%, or more identical to a CDR of a variable lightchain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18),1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24) (also shown in Table2).

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include one or more complementarity determiningregions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of the antibody produced by clone 3D8 (SEQ IDNOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15), and avariable light chain region of the antibodies or antigen bindingportions thereof can include one or more CDRs that are at least 80%,85%, 90%, 95%, 99%, or more identical to a CDR of a variable light chainregion of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11(SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24).

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include three CDRs that are at least 80%, 85%, 90%,95%, or 99%, or more identical to a CDR of a variable heavy chain regionof the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ IDNOs:10-12), or 3H2 (SEQ ID NOs:13-15).

In some embodiments, a variable light chain region of the antibodies orantigen binding portions thereof includes three CDRs that are at least80%, 85%, 90%, 95%, 99%, or more identical to a CDR of a variable lightchain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18),1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24).

In some embodiments, a variable light chain region of the antibodies orantigen binding portions thereof includes one or more CDRs that are atleast 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of avariable light chain region of the antibody produced by clone 3D8 (SEQID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24), and avariable heavy chain region of the antibodies or antigen bindingportions thereof includes three CDRs that are at least 80%, 85%, 90%,95%, or 99%, or more identical to a CDR of a variable heavy chain regionof the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ IDNOs:10-12), or 3H2 (SEQ ID NOs:13-15). The variable light chain regioncan include three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, ormore identical to a CDR of a variable light chain region of the antibodyproduced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or3H2 (SEQ ID NOs:22-24).

In certain embodiments, a variable heavy chain region of the antibodiesor antigen binding portions thereof includes three CDRs that areidentical to a CDR of a variable heavy chain region of the antibodyproduced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2(SEQ ID NOs:13-15), and a variable light chain region of the antibodiesor antigen binding portions thereof includes three CDRs that areidentical to a CDR of a variable light chain region of the antibodyproduced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or3H2 (SEQ ID NOs:22-24), e.g., a variable light chain region and variableheavy chain region of the antibody or antigen binding portion thereofare identical to a variable light chain region and variable heavy chainregion of the antibody produced by clone 3D8 (SEQ ID NO:1, SEQ ID NO:4),1B11 (SEQ ID NO:2, SEQ ID NO:5), or 3H2 (SEQ ID NO:3, SEQ ID NO:6).

In some embodiments, the antibodies or antigen binding portions thereofneutralize toxin B in vitro, inhibit binding of toxin B to mammaliancells, and/or neutralize toxin B in vivo.

In some embodiments, the antibodies or antigen binding portions thereofspecifically bind to an epitope in a C-terminal portion of toxin B(e.g., between amino acids 1777-2366 of toxin B). Other particularantibodies or antigen binding portions thereof, can specifically bind toan epitope within amino acid residues 1-100, 100-200, 200-300, 300-400,400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300,1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200,2100-2200 or 2200-2366 of toxin B, or any interval, portion or rangethereof.

In certain embodiments, the antibodies or antigen binding portionsthereof specifically bind to toxin B with a K_(D) of less than about20×10⁻⁶ M. In a particular embodiment, the antibody, or antigen bindingportion thereof, specifically binds to toxin B with a K_(D) of less thanabout 10×10⁻⁷ M, less than about 10×10⁻⁸ M, less than about 10×10⁻⁹ M,or less than about 10×10⁻¹⁰ M. In other particular embodiments, theantibody, or antigen binding portion thereof, specifically binds totoxin B with a K_(D) of less than about 50×10⁻¹⁰ M, less than about20×10⁻¹⁰ M, less than about 15×10⁻¹⁰ M, less than about 8×10⁻¹⁰ M, orless than about 5×10⁻¹⁰ M.

In various other embodiments, the antibodies or antigen binding portionsthereof include a variable heavy chain region including an amino acidsequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or moreidentical to a variable heavy chain region amino acid sequence of theantibody produced by clone 124-152 (i.e., the amino acid sequence shownin SEQ ID NO:54), 2A11, or 1 G10.

In certain embodiments, the antibodies or antigen binding portionsthereof include a variable light chain region comprising an amino acidsequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or moreidentical to a variable heavy chain region amino acid sequence of theantibody produced by clone 124-152 (i.e., the amino acid sequence shownin SEQ ID NO:58), 2A11, or 1 G10.

In certain embodiments, the antibodies or antigen binding portionsthereof each include both a variable heavy chain region including anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or moreidentical to a variable heavy chain region amino acid sequence of theantibody produced by clone 124-152 (i.e., the amino acid sequence shownin SEQ ID NO:54), 2A11, or 1 G10, and a variable light chain regionincluding an amino acid sequence that is at least 80%, 85%, 90%, 95%,98%, 99%, or more identical to a variable light chain amino acidsequence of the antibody produced by clone 124-152 (i.e., the amino acidsequence shown in SEQ ID NO:58), 2A11, or 1 G10.

In various embodiments, the antibodies or antigen binding portionsthereof specifically bind to an epitope that overlaps with an epitopebound by an antibody produced by clone 124-152, 2A11, or 1 G10 and/orcompete for binding to toxin B with an antibody produced by clone124-152, 2A11, or 1 G10.

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include one or more complementarity determiningregions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of the antibody produced by clone 124-152 (SEQ IDNOs: 62, 64, or 66), 2A11, or 1 G10 (Table 3).

A variable light chain region of the antibodies or antigen bindingportions thereof can include one or more complementarity determiningregions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of the antibody produced by clone 124-152 (SEQ IDNOs: 68, 70, or 72), 2A11, or 1 G10 (Table 4).

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include one or more complementarity determiningregions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of the antibody produced by clone 124-152 (SEQ IDNOs: 62, 64, or 66), 2A11, or 1 G10, and a variable light chain regionof the antibodies or antigen binding portions thereof can include one ormore CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identicalto a CDR of a variable light chain region of the antibody produced byclone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10.

A variable heavy chain region of the antibodies or antigen bindingportions thereof can include three CDRs that are at least 80%, 85%, 90%,95%, or 99%, or more identical to a CDR of a variable heavy chain regionof the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66),2A11, or 1 G10.

In certain embodiments, the variable light chain region of theantibodies or antigen binding portions thereof includes three CDRs thatare at least 80%, 85%, 90%, 95%, 99%, or more identical to a CDR of avariable light chain region of the antibody produced by clone 124-152(SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10.

In other embodiments, the variable light chain region of the antibodiesor antigen binding portions thereof includes one or more CDRs that areat least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of avariable light chain region of the antibody produced by clone 124-152(SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10, and a variable heavy chainregion of the antibodies or antigen binding portions thereof includesthree CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or moreidentical to a CDR of a variable heavy chain region of the antibodyproduced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10.The variable light chain region can include three CDRs that are at least80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variablelight chain region of the antibody produced by clone 124-152 (SEQ IDNOs: 68, 70, or 72), 2A11, or 1 G10.

In still other embodiments, the variable heavy chain region of theantibodies or antigen binding portions thereof includes three CDRs thatare identical to a CDR of a variable heavy chain region of the antibodyproduced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10,and a variable light chain region of the antibodies or antigen bindingportions thereof includes three CDRs that are identical to a CDR of avariable light chain region of the antibody produced by clone 124-152(SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10, e.g., a variable lightchain region and variable heavy chain region of the antibody or antigenbinding portion thereof are identical to a variable light chain regionand variable heavy chain region of the antibody produced by clone124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10.

The antibodies or antigen binding portions thereof can be full-lengthantibodies, can include an effector domain, e.g., an Fc domain, can beimmunoglobulin gamma isotype antibodies, single-chain antibodies, or Fabfragments. The antibodies or antigen binding portions thereof canfurther include a pharmaceutically acceptable carrier and/or a label.

In various embodiments, compositions including the antibodies or antigenbinding portions thereof are free of other human polypeptides (e.g.,they contain less than 5% human polypeptides other than the antibodiesor antigen binding portions thereof).

In yet another aspect, the invention features compositions including:(a) an isolated human monoclonal antibody or antigen binding portionthereof that specifically binds to an exotoxin of C. difficile; and (b)a polyclonal antibody or antigen binding portion thereof thatspecifically binds to an exotoxin of C. difficile.

In one embodiment, the human monoclonal antibody or antigen bindingportion thereof specifically binds to C. difficile toxin A, and thepolyclonal antibody or antigen binding portion thereof specificallybinds to C. difficile toxin B. In one embodiment, the human monoclonalantibody or antigen binding portion thereof specifically binds to C.difficile toxin B, and the polyclonal antibody or antigen bindingportion thereof specifically binds to C. difficile toxin A. Theantibodies can include other features described herein.

In another aspect, the invention features isolated human monoclonalantibodies or antigen binding portions thereof that specifically bind toan exotoxin of Clostridium difficile (C. difficile), wherein theantibodies: (a) include a heavy chain variable region that is theproduct of or derived from a human VH 3-33 gene; and/or (b) include alight chain variable region that is the product of or derived from ahuman Vκ gene selected from the group consisting of Vκ L19, Vκ L6 and VκL15. The antibodies or antigen binding portions thereof can includeother features described herein.

In another aspect, the invention features isolated human monoclonalantibodies or antigen binding portions thereof that specifically bind toan exotoxin of Clostridium difficile (C. difficile), wherein theantibodies: (a) include a heavy chain variable region that is theproduct of or derived from a human VH 5-51 gene; and/or (b) include alight chain variable region that is the product of or derived from ahuman Vκ A27 gene. The antibodies or antigen binding portions thereofalso can include other features described herein.

In another aspect, the invention features isolated polypeptides thatinclude an antigen binding portion of an antibody produced by hybridomaclone 3D8, 1B11, or 3H2 (also referred to herein as “3D8”, “1B11”, and“3H2”).

In another aspect, the invention features isolated polypeptides thatinclude an antigen binding portion of an antibody produced by hybridomaclone 124-152, 2A11, or 1 G10 (also referred to herein as “124-152”,“2A11”, and “1 G10”).

In another aspect, the invention features isolated monoclonal antibodiesor antigen binding portions thereof that specifically bind to anexotoxin of C. difficile, neutralize the toxin, inhibit, and/or protectfrom C. difficile-mediated disease. In one embodiment, the antibodies orantigen binding portions thereof are mammalian (e.g., human) antibodiesor antigen binding portions thereof. The antibodies or antigen bindingportions thereof can include other features described herein.

In another aspect, the invention features compositions including: (a) anisolated human monoclonal antibody or antigen binding portion thereofthat specifically binds to C. difficile toxin A; and (b) an isolatedhuman monoclonal antibody or antigen binding portion thereof thatspecifically binds to C. difficile toxin B.

In another aspect, the invention features isolated nucleic acidsincluding a sequence encoding polypeptides at least 75%, 80%, 85%, 90%,95%, 99%, or more identical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g.,wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%,99%, or more identical to SEQ ID NOs:38, 39, 40, 35, 36, or 37. Theinvention also features expression vectors including a nucleic acidencoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, or moreidentical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g., wherein the nucleicacid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or moreidentical to SEQ ID NOs:38, 39, 40, 35, 36, or 37, as well as hostcells, e.g., bacterial cells, e.g., E. coli cells, including a nucleicacid encoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, ormore identical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g., wherein thenucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or moreidentical to SEQ ID NOs:38, 39, 40, 35, 36, or 37.

In another aspect, the invention features isolated nucleic acidsincluding a sequence encoding a polypeptide that is at least 75%, 80%,85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 54, 56, 58, or 60,for example, wherein the nucleic acid sequence is at least 75%, 80%,85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 55, 57, 59, or 61.The invention also features expression vectors including a nucleic acidencoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, or moreidentical to SEQ ID NOs: 54, 56, 58, or 60, for example, wherein thenucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or moreidentical to SEQ ID NOs: 55, 57, 59, or 61. The invention also provideshost cells, e.g., bacterial cells, e.g., E. coli cells, that include anucleic acid encoding a polypeptide that is at least 75%, 80%, 85%, 90%,95%, 99%, or more identical to SEQ ID NOs: 54, 56, 58, or 60, forexample, wherein the nucleic acid sequence is at least 75%, 80%, 85%,90%, 95%, 99%, or more identical to SEQ ID NOs: 55, 57, 59, or 61.

The host cells can also be eukaryotic cells, e.g., yeast cells,mammalian cells, e.g., Chinese hamster ovary (CHO) cells, NSO cells, ormyeloma cells.

In another aspect, the invention features kits including an isolatedhuman monoclonal antibody or antigen binding portion thereof thatspecifically binds to an exotoxin of Clostridium difficile (C.difficile), e.g., an antibody or antigen binding portion thereofdescribed herein. The kit can include instructions for use in preventingor treating C. difficile-mediated disease.

The kit can further include a polyclonal antibody or antigen bindingportion thereof that specifically binds an exotoxin of C. difficile. Inone embodiment, the human monoclonal antibody or antigen binding portionthereof specifically binds to C. difficile toxin A. In one embodiment,the polyclonal antibody or antigen binding portion thereof specificallybinds to C. difficile toxin B.

In another aspect, the invention features kits including: (a) anisolated human monoclonal antibody that specifically binds to C.difficile toxin A; and (b) an isolated human monoclonal antibody thatspecifically binds to C. difficile toxin B.

The invention also features methods of treating C. difficile disease ina subject by administering to the subject an isolated human monoclonalantibody or antigen binding portion thereof that specifically binds toan exotoxin of Clostridium difficile (C. difficile) in an amounteffective to inhibit C. difficile disease, e.g., C. difficile-mediatedcolitis, antibiotic-associated colitis, C. difficile-mediatedpseudomembranous colitis (PMC), or diarrhea, or relapse of C.difficile-mediated disease. The antibody or antigen binding portionthereof can be administered, e.g., intravenously, intramuscularly, orsubcutaneously, to the subject.

The antibody or antigen binding portion thereof can be administeredalone or in combination with another therapeutic agent, e.g., a secondhuman monoclonal antibody or antigen binding portion thereof. In oneexample, the antibody or antigen binding portion thereof specificallybinds to C. difficile toxin A, and the second human monoclonal antibodyor antigen binding portion thereof specifically binds to C. difficiletoxin B. In another example, the second agent is an antibiotic, e.g.,vancomycin or metronidazole. The second agent can be polyclonalgamma-globulin (e.g., human gamma-globulin).

In a particular embodiment, an antibody or antigen binding portionthereof is administered which includes a variable light chain region anda variable heavy chain region identical to the variable light chainregion and variable heavy chain region of the antibody produced by clone3D8 (i.e., including a variable light chain region sequence identical toSEQ ID NO:4 and a variable heavy chain region sequence identical to SEQID NO:1.

In another embodiment, this antibody or antigen binding portion thereofis administered in combination with an antibody or antigen bindingportion thereof which includes a variable light chain region and avariable heavy chain region identical to the variable light chain regionand variable heavy chain region of the antibody produced by clone124-152 (i.e., including a variable light chain region sequenceidentical to SEQ ID NO:58 and a variable heavy chain region sequenceidentical to SEQ ID NO:54).

In yet another embodiment, an antibody or antigen binding portionproduced by clone 3D8 (i.e., including a variable light chain regionsequence identical to SEQ ID NO:4 and a variable heavy chain regionsequence identical to SEQ ID NO:1), is administered in combination withan antibody or antigen binding portion thereof produced by clone 124-152(i.e., including a variable light chain region sequence identical to SEQID NO:58 and a variable heavy chain region sequence identical to SEQ IDNO:54).

In another aspect, the invention features methods for making an antibodyor antigen binding portion thereof that specifically binds to anexotoxin of C. difficile, by immunizing a transgenic non-human animalhaving a genome comprising a human heavy chain transgene and a humanlight chain transgene with a composition that includes an inactivatedexotoxin, and isolating an antibody from the animal. The exotoxin can beinactivated, for example, by treatment with UDP-dialdehyde or bymutation (e.g., using recombinant methods). The method can furtherinclude evaluating binding of the antibody to the exotoxin.

The invention also features methods for making a human monoclonalantibody or antigen binding portion thereof by providing a nucleic acidencoding a human monoclonal antibody or antigen binding portion thereofthat specifically binds to an exotoxin of C. difficile, and expressingthe nucleic acid in a host cell.

In yet another aspect, the invention features a hybridoma ortransfectoma including a nucleic acid encoding antigen binding portions(e.g., CDRs, or variable regions) of the antibody produced by clone 3D8,1B11, or 3H2.

In yet another aspect, the invention features a hybridoma ortransfectoma including a nucleic acid encoding antigen binding portions(e.g., CDRs, or variable regions) of the antibody produced by clone124-152, 2A11, or 1 G10.

In addition, the invention features a method for making a hybridoma thatexpresses an antibody that specifically binds to an exotoxin of C.difficile by immunizing a transgenic non-human animal having a genomethat includes a human heavy chain transgene and a human light chaintransgene, with a composition that includes the exotoxin, wherein thetoxin is inactivated; isolating splenocytes from the animal; generatinghybridomas from the splenocytes; and selecting a hybridoma that producesan antibody that specifically binds to the exotoxin.

Treatment of humans with human monoclonal antibodies offers severaladvantages. For example, the antibodies are likely to be lessimmunogenic in humans than non-human antibodies. The therapy is rapid;toxin inactivation can occur as soon as the antibody reaches sites ofinfection and directly neutralizes the disease-causing toxin(s). Humanantibodies localize to appropriate sites in humans more efficiently thannon-human antibodies. Furthermore, the treatment is specific for C.difficile, and is unlikely to disrupt normal gut flora, unliketraditional antibiotic therapies.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table listing the amino acid sequences of the VH and VLchains encoded by mRNA sequences from each clone. Lowercase lettersrepresent amino acids in the leader peptide. CDRs are underlined. Clone3D8, which expresses 6 unique light chain V regions, only expressed thegroup I amino acid sequence.

FIG. 2A is a representation of the amino acid and nucleic acid sequencesof the VL chain expressed by clone 3D8. The V-segment and J-segmentgenes are listed above the amino acid and nucleic acid sequences. TheCDRs are overlined.

FIG. 2B is a representation of the amino acid and nucleic acid sequencesof the VH chain expressed by clone 3D8. The V-segment, D-segment andJ-segment genes are listed above the amino acid and nucleic acidsequences. The CDRs are overlined.

FIG. 3A is a representation of the amino acid and nucleic acid sequencesof the VL chain expressed by clone 1B11. The V-segment and J-segmentgenes are listed above the amino acid and nucleic acid sequences. TheCDRs are overlined.

FIG. 3B is a representation of the amino acid and nucleic acid sequencesof the VH chain expressed by clone 1B11. The V-segment, D-segment, andJ-segment genes are listed above the amino acid and nucleic acidsequences. The CDRs are overlined.

FIG. 4A is a representation of the amino acid and nucleic acid sequencesof the VL chain expressed by clone 33.3H2 (referred to herein as 3H2;33.3H2 and 3H2 are used interchangeably herein). The V-segment andJ-segment genes are listed above the amino acid and nucleic acidsequences. The CDRs are overlined.

FIG. 4B is a representation of the amino acid and nucleic acid sequencesof the VH chain expressed by clone 33.3H2. The V-segment and J-segmentgenes are listed above the amino acid and nucleic acid sequences. TheCDRs are overlined.

FIG. 5 is a graph depicting the results of ELISA assays, which measuredbinding of anti-toxin A monoclonal antibodies to toxin A.

FIG. 6A and FIG. 6B are a set of graphs depicting results of in vitroneutralization assays in the presence and absence of anti-toxin Amonoclonal antibodies. FIG. 6A depicts results for assays performed withIMR-90 cells. FIG. 6B depicts results for assays performed with T-84cells.

FIG. 7 is a schematic representation of the toxin A polypeptide,indicating fragments that were analyzed for epitope mapping studies.

FIG. 8A and FIG. 8B are schematic representations of toxin A fragmentsanalyzed for epitope mapping studies.

FIG. 9 is a table listing the results of in vivo assays to determinemouse protection from lethal challenge with toxin A by anti-toxin Amonoclonal antibodies.

FIG. 10 is a graph depicting the results of mouse ileal loop fluidaccumulation assays to measure efficacy of anti-toxin antibodyneutralization in vivo.

FIG. 11A is a schematic diagram of the timeline of administration ofvarious agents to hamsters in a hamster relapse model.

FIG. 11B is a graph depicting the results of the assays as thepercentage of hamsters surviving clindamycin treatment followed by C.difficile challenge.

FIG. 12 is a graph depicting results of hamster relapse assays as thepercentage of hamsters surviving clindamycin treatment followed by C.difficile challenge.

FIG. 13 is a graph depicting results of assays in which in vitroneutralization of toxin A and toxin B was measured in the presence andabsence of polyclonal antisera from goats immunized with toxoid B.“G330” refers to samples in which sera from goat #330 were tested.“G331” refers to samples in which sera from goat #331 were tested.

FIG. 14 is a schematic diagram of the timeline of administration ofvarious agents to hamsters in a hamster relapse model.

FIG. 15 is a graph depicting the results of hamster relapse assays asthe percentage of hamsters surviving clindamycin treatment followed byC. difficile challenge. Hamsters were treated with vancomycin,vancomycin and 3D8, vancomycin and antisera from goat #331, orvancomycin, 3D8, and antisera from goat #331.

FIG. 16 is a graph depicting the results of hamster relapse assays asthe percentage of healthy animals after clindamycin treatment followedby C. difficile challenge. “Goat 331” refers to antisera from goat #331.

FIG. 17 is a graph depicting the results of hamster relapse assays asthe percentage of hamsters surviving clindamycin treatment followed byC. difficile challenge. Hamsters were immunized with a fragment of toxinB prior to clindamycin treatment. Hamsters were treated with vancomycin,vancomycin and 3D8, or received no treatment.

FIG. 18 is a graph depicting the results of hamster relapse assays asthe percentage of healthy animals after clindamycin treatment followedby C. difficile challenge. Hamsters were immunized with a fragment oftoxin B prior to clindamycin treatment.

FIG. 19 is a schematic diagram of the timeline of administration ofvarious agents to hamsters in a C. difficile direct challenge model.“331” refers to antisera from goat #331. “Clinda” refers to treatmentwith clindamycin.

FIG. 20 is a graph depicting the results of direct challenge assays asthe percentage of hamsters surviving direct C. difficile challenge.

FIG. 21 is a graph depicting the results of direct challenge assays asthe percentage of healthy animals after direct challenge with C.difficile.

FIG. 22 is a representation of the amino acid sequence of C. difficiletoxin A.

FIG. 23 is a representation of the amino acid sequence of C. difficiletoxin B.

FIG. 24 is a graph depicting the results of primary challenge assays asthe percentage of hamsters surviving direct C. difficile challenge.

FIG. 25 is a graph depicting the results of primary challenge assays asthe percentage of hamsters surviving direct C. difficile challenge.

FIG. 26 is a graph depicting the results of primary challenge assays asthe percentage of hamsters surviving direct C. difficile challenge.

FIG. 27 is a graph depicting results of assays in which in vitroneutralization of toxin A and toxin B was measured in the presence ofmonoclonal antibodies to toxin B or goat polyclonal sera against toxinB.

FIG. 28 is a representation of the amino acid and nucleic acid sequencesof the VH chain expressed by clone 124-152. The V-segment, D-segment andJ-segment genes are listed above the amino acid and nucleic acidsequences. The CDRs are overlined.

FIG. 29 is a representation of the amino acid and nucleic acid sequencesof the VL chain expressed by clone 124-152. The V-segment and J-segmentgenes are listed above the amino acid and nucleic acid sequences. TheCDRs are overlined.

FIG. 30 is a representation of the amino acid and related germlinesequence of the VH chain expressed by clone 124-152. The V-segment,D-segment and J-segment genes are listed above the amino acid sequences.The CDRs are overlined.

FIG. 31 is a representation of the amino acid and related germlinesequences of the VL chain expressed by clone 124-152. The V-segment andJ-segment genes are listed above the amino acid sequences. The CDRs areoverlined.

FIG. 32 is a schematic representation of the toxin B polypeptide,indicating fragments that were analyzed for epitope mapping studies.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear understanding of the specification andclaims, the following definitions are conveniently provided below.

Definitions

The term “toxin A” refers to the toxin A protein encoded by C.difficile. The amino acid sequence of C. difficile toxin A (SEQ IDNO:41) is provided in GenBank® under accession number A37052, version GI98593 (see also FIG. 22). “Toxin B” refers to the toxin B proteinencoded by C. difficile. The amino acid sequence of C. difficile toxin B(SEQ ID NO: 42) is provided in GenBank® under accession number S70172,version GI 7476000 (see also FIG. 23). “Protein” is used interchangeablywith “polypeptide.”

An “anti-C. difficile antibody” is an antibody that interacts with(e.g., binds to) a protein or other component produced by C. difficilebacteria. An “anti-toxin antibody” is an antibody that interacts with atoxin produced by C. difficile (e.g., toxin A or toxin B). An anti-toxinprotein antibody may bind to an epitope, e.g., a conformational or alinear epitope, or to a fragment of the full-length toxin protein.

A “human antibody,” is an antibody that has variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies described herein may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo).

An anti-toxin antibody, or antigen binding portion thereof, can beadministered alone or in combination with a second agent. The subjectcan be a patient infected with C. difficile, or having a symptom of C.difficile-associated disease (“CDAD”; e.g., diarrhea, colitis, abdominalpain) or a predisposition towards C. difficile-associated disease (e.g.,undergoing treatment with antibiotics, or having experienced C.difficile-associated disease and at risk for relapse of the disease).The treatment can be to cure, heal, alleviate, relieve, alter, remedy,ameliorate, palliate, improve, or affect the infection and the diseaseassociated with the infection, the symptoms of the disease, or thepredisposition toward the disease.

An amount of an anti-toxin antibody effective to treat a CDAD, or a“therapeutically effective amount,” is an amount of the antibody that iseffective, upon single or multiple dose administration to a subject, ininhibiting CDAD in a subject. A therapeutically effective amount of theantibody or antibody fragment may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion isoutweighed by the therapeutically beneficial effects. The ability of anantibody to inhibit a measurable parameter can be evaluated in an animalmodel system predictive of efficacy in humans. For example, the abilityof an anti-toxin antibody to protect mice from lethal challenge with C.difficile can predict efficacy in humans. Other animal models predictiveof efficacy are described herein, such as the intestinal ligation modeldescribed in the Examples. Alternatively, this property of an antibodyor antibody composition can be evaluated by examining the ability of thecompound to modulate, such modulation in vitro by assays known to theskilled practitioner. In vitro assays include binding assays, such asELISA, and neutralization assays.

An amount of an anti-toxin antibody effective to prevent a disorder, ora “a prophylactically effective amount,” of the antibody is an amountthat is effective, upon single- or multiple-dose administration to thesubject, in preventing or delaying the occurrence of the onset orrecurrence of CDAD, or inhibiting a symptom thereof. However, if longertime intervals of protection are desired, increased doses can beadministered.

The terms “agonize,” “induce,” “inhibit,” “potentiate,” “elevate,”“increase,” “decrease,” or the like, e.g., which denote quantitativedifferences between two states, refer to a difference, e.g., astatistically or clinically significant difference, between the twostates.

As used herein, “specific binding” or “specifically binds to” refers tothe ability of an antibody to: (1) bind to a toxin of C. difficile withan affinity of at least 1×10⁷ M⁻¹, and (2) bind to a toxin of C.difficile with an affinity that is at least two-fold greater than itsaffinity for a nonspecific antigen.

An “antibody” is a protein including at least one or two, heavy (H)chain variable regions (abbreviated herein as VHC), and at least one ortwo light (L) chain variable regions (abbreviated herein as VLC). TheVHC and VLC regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, 1991, and Chothia, C. etal., J. Mol. Biol. 196:901-917, 1987, which are incorporated herein byreference). Preferably, each VHC and VLC is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VHC or VLC chain of the antibody can further include all or part ofa heavy or light chain constant region. In one embodiment, the antibodyis a tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains, wherein the heavy and light immunoglobulin chainsare inter-connected by, e.g., disulfide bonds. The heavy chain constantregion includes three domains, CH1, CH2 and CH3. The light chainconstant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts withan antigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof), wherein the light chains of theimmunoglobulin may be of types kappa or lambda.

“Immunoglobulin” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes. Therecognized human immunoglobulin genes include the kappa, lambda, alpha(IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Full-length immunoglobulin “light chains” (about 25 KD and214 amino acids) are encoded by a variable region gene at theNH₂-terminus (about 110 amino acids) and a kappa or lambda constantregion gene at the COOH-terminus. Full-length immunoglobulin “heavychains” (about 50 KD and 446 amino acids), are similarly encoded by avariable region gene (about 116 amino acids) and one of the otheraforementioned constant region genes, e.g., gamma (encoding about 330amino acids). The term “immunoglobulin” includes an immunoglobulinhaving: CDRs from a human or non-human source. The framework of theimmunoglobulin can be human, humanized, or non-human, e.g., a murineframework modified to decrease antigenicity in humans, or a syntheticframework, e.g., a consensus sequence.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG₁) that is encoded by heavy chain constant region genes.

The term “antigen binding portion” of an antibody (or simply “antibodyportion,” or “portion”), as used herein, refers to a portion of anantibody that specifically binds to a toxin of C. difficile (e.g., toxinA), e.g., a molecule in which one or more immunoglobulin chains is notfull length, but which specifically binds to a toxin. Examples ofbinding portions encompassed within the term “antigen-binding portion”of an antibody include (i) a Fab fragment, a monovalent fragmentconsisting of the VLC, VHC, CL and CH1 domains; (ii) a F(ab′)₂ fragment,a bivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the VHCand CH1 domains; (iv) a Fv fragment consisting of the VLC and VHCdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341:544-546, 1989), which consists of a VHC domain; and (vi) anisolated complementarity determining region (CDR) having sufficientframework to specifically bind, e.g., an antigen binding portion of avariable region. An antigen binding portion of a light chain variableregion and an antigen binding portion of a heavy chain variable region,e.g., the two domains of the Fv fragment, VLC and VHC, can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the VLC and VHC regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso encompassed within the term “antigen binding portion” of anantibody. These antibody portions are obtained using conventionaltechniques known to those with skill in the art, and the portions arescreened for utility in the same manner as are intact antibodies.

The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or portions thereof with a single molecular composition.

The term “recombinant” antibody, as used herein, refers to antibodiesthat are prepared, expressed, created, or isolated by recombinant means,such as antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes orantibodies prepared, expressed, created, or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant antibodies include humanized, CDRgrafted, chimeric, in vitro generated (e.g., by phage display)antibodies, and may optionally include constant regions derived fromhuman germline immunoglobulin sequences.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) refers to a first amino acid or nucleotide sequence thatcontains a sufficient number of identical or equivalent (e.g., with asimilar side chain, e.g., conserved amino acid substitutions) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences havesimilar activities. In the case of antibodies, the second antibody hasthe same specificity and has at least 50% of the affinity of the firstantibody.

Calculations of “homology” between two sequences are performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 50% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. The percent homology between two amino acid sequences isdetermined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453,1970, algorithm which has been incorporated into the GAP program in theGCG software package, using a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which isincorporated herein by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: 1) lowstringency hybridization conditions: 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); 2) medium stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridizationconditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C.

It is understood that the antibodies and antigen binding portionsthereof described herein may have additional conservative ornon-essential amino acid substitutions, which do not have a substantialeffect on the polypeptide functions. Whether or not a particularsubstitution will be tolerated, i.e., will not adversely affect desiredbiological properties, such as binding activity, can be determined asdescribed in Bowie et al., Science, 247:1306-1310, 1990. A “conservativeamino acid substitution” is one in which an amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide, such as a binding agent,e.g., an antibody, without substantially altering a biological activity,whereas an “essential” amino acid residue results in such a change.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Overview

C. difficile is a gram positive, toxin-producing bacterium that causesantibiotic-associated diarrhea and colitis in humans. Provided hereinare methods and compositions for treatment and prevention of C.difficile-associated disease (CDAD). The compositions include antibodiesthat recognize proteins and other molecular components (e.g., lipids,carbohydrates, nucleic acids) of C. difficile bacteria, includingantibodies that recognize toxins produced by C. difficile (e.g., toxin Aand toxin B). In particular, human monoclonal antibodies are provided.In certain embodiments, these human monoclonal antibodies are producedin mice expressing human immunoglobulin gene segments (described below).Combinations of anti-toxin antibodies are also provided.

The new methods include administering antibodies (and antigen-bindingportions thereof) that bind to a C. difficile toxin to a subject toinhibit CDAD in the subject. For example, human monoclonal anti-toxin Aantibodies described herein can neutralize toxin A and inhibit relapseof C. difficile-mediated disease. In other examples, combinations ofanti-toxin A antibodies (e.g., anti-toxin A monoclonal antibodies) andanti-toxin B antibodies can be administered to inhibit primary diseaseand reduce the incidence of disease relapse. The human monoclonalantibodies may localize to sites of disease (e.g., the gut) in vivo.

1. Generation of Antibodies Immunogens

In general, animals are immunized with antigens expressed by C.difficile to produce antibodies. For producing anti-toxin antibodies,animals are immunized with inactivated toxins, or toxoids. Toxins can beinactivated, e.g., by treatment with formaldehyde, glutaraldehyde,peroxide, or oxygen treatment (see, e.g., Relyveld et al., Methods inEnzymology 93:24, 1983; Woodrow and Levine, eds., New GenerationVaccines, Marcel Dekker, Inc., New York, 1990). Mutant C. difficiletoxins with reduced toxicity can be produced using recombinant methods(see, e.g., U.S. Pat. Nos. 5,085,862; 5,221,618; 5,244,657; 5,332,583;5,358,868; and 5,433,945). For example, mutants containing deletions orpoint mutations in the toxin active site can be made. Recombinantfragments of the toxins can be used as immunogens. Another approach isto inactivate the toxin by treatment with UDP-dialdehyde (Genth et al.,Inf. and Immun., 68(3):1094-1101, 2000). This method preserves thenative structure of the toxin more readily than other treatments, andthus can elicit antibodies more reactive to the native toxin. Thismethod is also described in Example 1, below.

Anti-toxin antibodies that bind and neutralize toxin A can interact withspecific epitopes of toxin A. For example, an anti-toxin A antibody canbind an epitope in an N-terminal region of toxin A (e.g., between aminoacids 1-1033 of toxin A), or a C-terminal region (e.g., between aminoacids 1853-2710 of toxin A). In one example, an antibody that binds andneutralizes toxin A binds to an epitope within amino acids 1853-2710 oftoxin A.

Similarly, anti-toxin B antibodies can recognize a specific epitope oftoxin B, e.g., an N-terminal epitope, or a C-terminal epitope. In oneexample, an antibody that binds and neutralizes toxin B binds to anepitope within amino acids 1777-2366 of toxin B.

Generation of Human Monoclonal Antibodies in HuMAb Mice

Monoclonal antibodies can be produced in a manner not possible withpolyclonal antibodies. Polyclonal antisera vary from animal to animal,whereas monoclonal preparations exhibit a uniform antigenic specificity.Murine animal systems are useful to generate monoclonal antibodies, andimmunization protocols, techniques for isolating and fusing splenocytes,and methods and reagents for producing hybridomas are well known.Monoclonal antibodies can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature, 256: 495, 1975. See generally, Harlow, E. and Lane, D.Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988.

Although these standard techniques are known, it is desirable to usehumanized or human antibodies rather than murine antibodies to treathuman subjects, because humans mount an immune response to antibodiesfrom mice and other species. The immune response to murine antibodies iscalled a human anti-mouse antibody or HAMA response (Schroff, R. et al.,Cancer Res., 45, 879-885, 1985) and is a condition that causes serumsickness in humans and results in rapid clearance of the murineantibodies from an individual's circulation. The immune response inhumans has been shown to be against both the variable and the constantregions of murine immunoglobulins. Human monoclonal antibodies are saferfor administration to humans than antibodies derived from other animalsand human polyclonal antibodies.

One useful type of animal in which to generate human monoclonalantibodies is a transgenic mouse that expresses human immunoglobulingenes rather than its own mouse immunoglobulin genes. Such transgenicmice, e.g., “HuMAb™” mice, contain human immunoglobulin gene minilocithat encode unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, N. etal., Nature 368(6474): 856-859, 1994, and U.S. Pat. No. 5,770,429).Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGic monoclonal antibodies (Lonberg, N. et al., supra;reviewed in Lonberg, N. Handbook of Experimental Pharmacology113:49-101, 1994; Lonberg, N. and Huszar, D., Intern. Rev. Immunol., 13:65-93, 1995, and Harding, F. and Lonberg, N., Ann. N.Y. Acad. Sci.,764:536-546, 1995).

The preparation of such transgenic mice is described in further detailin Taylor, L. et al., Nucleic Acids Research, 20:6287-6295, 1992; Chen,J. et al., International Immunology 5: 647-656, 1993; Tuaillon et al.,Proc. Natl. Acad. Sci., USA 90:3720-3724, 1993; Choi et al., NatureGenetics, 4:117-123, 1993; Chen, J. et al., EMBO J., 12: 821-830, 1993;Tuaillon et al., J. Immunol., 152:2912-2920, 1994; Taylor, L. et al.,International Immunology, 6: 579-591, 1994; and Fishwild, D. et al.,Nature Biotechnology, 14: 845-851, 1996. See further, U.S. Pat. Nos.5,545,806; 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,874,299 and 5,877,397, all by Lonberg and Kay,and PCT Publication Nos. WO 01/14424, WO 98/24884, WO 94/25585, WO93/1227, and WO 92/03918.

To generate fully human monoclonal antibodies to an antigen, HuMAb micecan be immunized with an immunogen, as described by Lonberg, N. et al.Nature, 368(6474): 856-859, 1994; Fishwild, D. et al., NatureBiotechnology, 14: 845-851, 1996 and WO 98/24884. Preferably, the micewill be 6-16 weeks of age upon the first immunization. For example, apurified preparation of inactivated toxin A can be used to immunize theHuMAb mice intraperitoneally. To generate antibodies against C.difficile proteins, lipids, and/or carbohydrate molecules, mice can beimmunized with killed or nonviable C. difficile organisms.

HuMAb transgenic mice respond best when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by IP immunizations every other week (up to a total of 6) withantigen in incomplete Freund's adjuvant. The immune response can bemonitored over the course of the immunization protocol with plasmasamples being obtained by retroorbital bleeds. The plasma can bescreened, for example by ELISA or flow cytometry, and mice withsufficient titers of anti-toxin human immunoglobulin can be used forfusions. Mice can be boosted intravenously with antigen 3 days beforesacrifice and removal of the spleen. It is expected that 2-3 fusions foreach antigen may need to be performed. Several mice are typicallyimmunized for each antigen.

The mouse splenocytes can be isolated and fused with PEG to a mousemyeloma cell line based upon standard protocols. The resultinghybridomas are then screened for the production of antigen-specificantibodies. For example, single cell suspensions of splenic lymphocytesfrom immunized mice are fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 20%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells are cultured in medium in which the HATis replaced with HT. Supernatants from individual wells are thenscreened by ELISA for human anti-toxin cell monoclonal IgM and IgGantibodies. The antibody secreting hybridomas are replated, screenedagain, and if still positive for human IgG, anti-toxin monoclonalantibodies, can be subcloned at least twice by limiting dilution. Thestable subclones are then cultured in vitro to generate small amounts ofantibody in tissue culture medium for characterization.

In one embodiment, the transgenic animal used to generate humanantibodies to the toxin contains at least one, typically 2-10, andsometimes 25-50 or more copies of the transgene described in Example 12of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animal containing asingle copy of a light chain transgene described in Examples 5, 6, 8, or14 of WO 98/24884, and the offspring bred with the J_(H) deleted animaldescribed in Example 10 of WO 98/24884, the contents of which are herebyexpressly incorporated by reference. Animals are bred to homozygosityfor each of these three traits. Such animals have the followinggenotype: a single copy (per haploid set of chromosomes) of a humanheavy chain unrearranged mini-locus (described in Example 12 of WO98/24884), a single copy (per haploid set of chromosomes) of arearranged human K light chain construct (described in Example 14 of WO98/24884), and a deletion at each endogenous mouse heavy chain locusthat removes all of the functional J_(H) segments (described in Example10 of WO 98/24884). Such animals are bred with mice that are homozygousfor the deletion of the J_(H) segments (Examples 10 of WO 98/24884) toproduce offspring that are homozygous for the J_(H) deletion andhemizygous for the human heavy and light chain constructs. The resultantanimals are injected with antigens and used for production of humanmonoclonal antibodies against these antigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regard to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Examples 9 and 12 of WO 98/24884.Furthermore, a substantial fraction of the B cells will be monospecificwith regards to the human or mouse light chains, because expression ofthe single copy of the rearranged human kappa light chain gene willallelically and isotypically exclude the rearrangement of the endogenousmouse kappa and lambda chain genes in a significant fraction of B-cells.

In one embodiment, the transgenic mouse will exhibit immunoglobulinproduction with a significant repertoire, ideally substantially similarto that of a native mouse. Thus, for example, in embodiments where theendogenous Ig genes have been inactivated, the total immunoglobulinlevels will range from about 0.1 to 10 mg/ml of serum, e.g., 0.5 to 5mg/ml, or at least about 1.0 mg/ml. When a transgene capable ofeffecting a switch to IgG from IgM has been introduced into thetransgenic mouse, the adult mouse ratio of serum IgG to IgM ispreferably about 10:1. The IgG to IgM ratio will be much lower in theimmature mouse. In general, greater than about 10%, e.g., about 40 to80% of the spleen and lymph node B cells will express exclusively humanIgG protein.

The repertoire in the transgenic mouse will ideally approximate thatshown in a non-transgenic mouse, usually at least about 10% as high,preferably 25 to 50% or more as high. Generally, at least about athousand different immunoglobulins (ideally IgG), preferably 10⁴ to 10⁶or more, will be produced, depending primarily on the number ofdifferent V, J, and D regions introduced into the mouse genome.Typically, the immunoglobulins will exhibit an affinity for preselectedantigens of at least about 10⁷M⁻¹, 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹, 10¹²M⁻¹, orgreater, e.g., up to 10¹³M⁻¹ or greater.

HuMAb mice can produce B cells that undergo class-switching viaintratransgene switch recombination (cis-switching) and expressimmunoglobulins reactive with the toxin. The immunoglobulins can behuman sequence antibodies, wherein the heavy and light chainpolypeptides are encoded by human transgene sequences, which may includesequences derived by somatic mutation and V region recombinatorialjoints, as well as germline-encoded sequences. These human sequenceimmunoglobulins can be referred to as being substantially identical to apolypeptide sequence encoded by a human VL or VH gene segment and ahuman JL or JL segment, even though other non-germline sequences may bepresent as a result of somatic mutation and differential V-J and V-D-Jrecombination joints. With respect to such human sequence antibodies,the variable regions of each chain are typically at least 80 percentencoded by human germline V, J, and, in the case of heavy chains, D,gene segments. Frequently at least 85 percent of the variable regionsare encoded by human germline sequences present on the transgene. Often90 or 95 percent or more of the variable region sequences are encoded byhuman germline sequences present on the transgene. However, sincenon-germline sequences are introduced by somatic mutation and VJ and VDJjoining, the human sequence antibodies will frequently have somevariable region sequences (and less frequently constant regionsequences) that are not encoded by human V, D, or J gene segments asfound in the human transgene(s) in the germline of the mice. Typically,such non-germline sequences (or individual nucleotide positions) willcluster in or near CDRs, or in regions where somatic mutations are knownto cluster.

The human sequence antibodies that bind to the toxin can result fromisotype switching, such that human antibodies comprising a humansequence gamma chain (such as gamma 1, gamma 2, or gamma 3) and a humansequence light chain (such as K) are produced. Such isotype-switchedhuman sequence antibodies often contain one or more somatic mutation(s),typically in the variable region and often in or within about 10residues of a CDR) as a result of affinity maturation and selection of Bcells by antigen, particularly subsequent to secondary (or subsequent)antigen challenge. These high affinity human sequence antibodies havebinding affinities of at least about 1×10⁹ M⁻¹, typically at least 5×10⁹M⁻¹, frequently more than 1×10¹⁰ M⁻¹, and sometimes 5×10¹⁰ M⁻¹ to 1×10¹¹M⁻¹ or greater.

Anti-toxin antibodies can also be raised in other mammals, includingnon-transgenic mice, humans, rabbits, and goats.

Anti-Toxin a Antibodies

Human monoclonal antibodies that specifically bind to toxin A includeantibodies produced by the 3D8, 1B11, and 3H2 clones described herein.Antibodies with variable heavy chain and variable light chain regionsthat are at least 80%, or more, identical to the variable heavy andlight chain regions of 3D8, 1B11, and 3H2 can also bind to toxin A. Inrelated embodiments, anti-toxin A antibodies include, for example, thecomplementarity determining regions (CDR) of variable heavy chainsand/or variable light chains of 3D8, 1B11, or 3H2. The CDRs of thevariable heavy chain regions from these clones are shown in Table 1,below.

TABLE 1 Variable Heavy Chain CDR Amino Acid Sequences Clone Chain CDRAmino Acid Sequence SEQ ID NO: 3D8 H CDR1 NYGMH 7 1B11 H CDR1 SYGMH 103H2 H CDR1 KYGMH 13 3D8 H CDR2 LIWYDGSNEDYTDSVKG 8 1B11 H CDR2VIWASGNKKYYIESVEG 11 3H2 H CDR2 VIWYDGTNKYYADSMKG 14 3D8 H CDR3WGMVRGVIDVFDI 9 1B11 H CDR3 ANFDY 12 3H2 H CDR3 DPPTANY 15

The CDRs of the variable light chain regions from these clones are shownin table 2, below.

TABLE 2 Variable Light Chain CDR Amino Acid Sequences Clone Chain CDRAmino Acid Sequence SEQ ID NO: 3D8 L CDR1 RASQGISSWLA 16 1B11 L CDR1RASQSVSSYLA 19 3H2 L CDR1 RASQGISSWLA 22 3D8 L CDR2 AASSLQS 17 1B11 LCDR2 DASNRAT 20 3H2 L CDR2 AASSLQS 23 3D8 L CDR3 QQANSFPWT 18 1B11 LCDR3 QQRSNWSQFT 21 3H2 L CDR3 QQYKSYPVT 24

CDRs are the portions of immunoglobulins that determine specificity fora particular antigen. In certain embodiments, CDRs corresponding to theCDRs in tables 1 and 2 having sequence variations (e.g., conservativesubstitutions) may bind to toxin A. For example, CDRs, in which 1, 2 3,4, or 5 residues, or less than 20% of total residues in the CDR, aresubstituted or deleted can be present in an antibody (or antigen bindingportion thereof) that binds toxin A.

Similarly, anti-toxin antibodies can have CDRs containing a consensussequence, as sequence motifs conserved amongst multiple antibodies canbe important for binding activity. For example, CDR1 of a variable lightchain region of the antibodies or antigen binding portions thereof caninclude the amino acid sequence R-A-S-Q-X-X-S-S-X-L-A (SEQ ID NO: 25),CDR2 of a variable light chain region of the antibodies or antigenbinding portions thereof can include the amino acid sequenceA-S-X-X-X-S/T (SEQ ID NO:26), and/or CDR3 of a variable light chainregion of the antibodies or antigen binding portions thereof can includethe amino acid sequence Q-Q-X-X-S/N-X-P/S (SEQ ID NO:27), wherein X isany amino acid.

In some embodiments, CDR1 of a variable heavy chain region of theantibodies or antigen binding portions thereof includes the amino acidsequence Y-G-M-H (SEQ ID NO:28), and/or CDR2 of a variable heavy chainregion of the antibodies or antigen binding portions thereof includesthe amino acid sequence I-W-X-X-G-X-X-X-Y-X-X-S-X-X-G (SEQ ID NO:29),wherein X is any amino acid.

Human anti-toxin antibodies can include variable regions that are theproduct of, or derived from, specific human immunoglobulin genes. Forexample, the antibodies can include a variable heavy chain region thatis the product of, or derived from a human VH3-33 gene. Numeroussequences for antibodies derived from this gene are available inGenBank® (see, e.g., Acc. No: AJ555951, GI No:29836865; Acc.No:AJ556080, GI No.:29837087; Acc. No.: AJ556038, GI No.:29837012, andother human VH3-33 rearranged gene segments provided in GenBank®). Theantibodies can also, or alternatively, include a light chain variableregion that is the product of, or derived from a human Vκ L19 gene (see,e.g., GenBank® Acc. No. AJ556049, GI No:29837033 for a partial sequenceof a rearranged human Vκ L19 gene segment). As known in the art, anddescribed in this section, above, variable immunoglobulin regions ofrecombined antibodies are derived by a process of recombination in vivoin which variability is introduced to genomic segments encoding theregions. Accordingly, variable regions derived from a human VH-33 or VκL19 gene can include nucleotides that are different that those in thegene found in non-lymphoid tissues. These nucleotide differences aretypically concentrated in the CDRs.

Anti-Toxin B Antibodies

Human monoclonal antibodies that specifically bind to toxin B includeantibodies produced by the 124-152, 2A11, and 1 G10 clones describedherein. Antibodies with variable heavy chain and variable light chainregions that are at least 80%, or more, identical to the variable heavyand light chain regions of −152, 2A11, and 1 G10 can also bind to toxinB. In related embodiments, anti-toxin B antibodies include, for example,the complementarity determining regions (CDR) of variable heavy chainsand/or variable light chains of −152, 2A11, or 1 G10. The CDRs of thevariable heavy chain regions from these clones are shown in Table 3,below.

TABLE 3 Variable Heavy Chain CDR Amino Acid Sequences SEQ ID NO: SEQ IDNO: Clone Chain CDR Amino Acid Sequence (a.a.) (n.t.) 124-152 H CDR1SYWIG 62 63 124-152 H CDR2 IFYPGDSSTRYSPSFQG 64 65 124-152 H CDR3RRNWGNAFDI 66 67

The CDRs of the variable light chain regions from these clones are shownin Table 4, below.

TABLE 4 Variable Light Chain CDR Amino Acid Sequences SEQ ID NO: SEQ IDNO: Clone Chain CDR Amino Acid Sequence (a.a.) (n.t.) 124-152 L CDR1RASQSVSSSYLAW 68 69 124-152 L CDR2 GASSRAT 70 71 124-152 L CDR3QQYGSSTWT 72 73

CDRs are the portions of immunoglobulins that determine specificity fora particular antigen. In certain embodiments, CDRs corresponding to theCDRs in Tables 3 and 4 having sequence variations (e.g., conservativesubstitutions) may bind to toxin B. For example, CDRs, in which 1, 2, 3,4, or 5 residues, or less than 20% of total residues in the CDR, aresubstituted or deleted can be present in an antibody (or antigen bindingportion thereof) that binds toxin B.

Human anti-toxin B antibodies can include variable regions that are theproduct of, or derived from, specific human immunoglobulin genes (seeFIGS. 28-31). For example, the antibodies can include a variable heavychain region that is the product of, or derived from a human VH 5-51gene. The antibodies can also, or alternatively, include a light chainvariable region that is the product of, or derived from a human Vκ A27gene and/or JK1 gene. As known in the art, and described in thissection, above, variable immunoglobulin regions of recombined antibodiesare derived by a process of recombination in vivo in which variabilityis introduced to genomic segments encoding the regions. Accordingly,variable regions derived from a human VH-5-51 or Vκ A27/JK1 gene caninclude nucleotides that are different that those in the gene found innon-lymphoid tissues. These nucleotide differences are typicallyconcentrated in the CDRs.

2. Production and Modification of Antibodies

Many different forms of anti-toxin antibodies can be useful in theinhibition of CDAD. The antibodies can be of the various isotypes,including: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, orIgE. Preferably, the antibody is an IgG isotype, e.g., IgG1. Theantibody molecules can be full-length (e.g., an IgG1 or IgG4 antibody)or can include only an antigen-binding fragment (e.g., a Fab, F(ab′)₂,Fv or a single chain Fv fragment). These include monoclonal antibodies(e.g., human monoclonal antibodies), recombinant antibodies, chimericantibodies, and humanized antibodies, as well as antigen-bindingportions of the foregoing.

Anti-toxin antibodies or portions thereof useful in the presentinvention can also be recombinant antibodies produced by host cellstransformed with DNA encoding immunoglobulin light and heavy chains of adesired antibody. Recombinant antibodies may be produced by knowngenetic engineering techniques. For example, recombinant antibodies canbe produced by cloning a nucleotide sequence, e.g., a cDNA or genomicDNA, encoding the immunoglobulin light and heavy chains of the desiredantibody. The nucleotide sequence encoding those polypeptides is theninserted into an expression vector so that both genes are operativelylinked to their own transcriptional and translational expression controlsequences. The expression vector and expression control sequences arechosen to be compatible with the expression host cell used. Typically,both genes are inserted into the same expression vector. Prokaryotic oreukaryotic host cells may be used.

Expression in eukaryotic host cells is preferred because such cells aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody. However, any antibodyproduced that is inactive due to improper folding can be renaturedaccording to well known methods (Kim and Baldwin, Ann. Rev. Biochem.,51:459-89, 1982). It is possible that the host cells will produceportions of intact antibodies, such as light chain dimers or heavy chaindimers, which also are antibody homologs according to the presentinvention.

The antibodies described herein also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S., Science, 229:1202, 1985). For example, in one embodiment,the gene(s) of interest, e.g., human antibody genes, can be ligated intoan expression vector such as a eukaryotic expression plasmid such asused in a GS gene expression system disclosed in WO 87/04462, WO89/01036 and EP 338 841, or in other expression systems well known inthe art. The purified plasmid with the cloned antibody genes can beintroduced in eukaryotic host cells such as CHO-cells or NSO-cells oralternatively other eukaryotic cells like a plant derived cells, fungior yeast cells. The method used to introduce these genes can be anymethod described in the art, such as electroporation, lipofectine,lipofectamine or ballistic transfection, in which cells are bombardedwith microparticles carrying the DNA of interest (Rodin, et al. Immunol.Lett., 74(3):197-200, 2000). After introducing these antibody genes inthe host cells, cells expressing the antibody can be identified andselected. These cells represent the transfectomas which can then beamplified for their expression level and upscaled to produce antibodies.Recombinant antibodies can be isolated and purified from these culturesupernatants and/or cells using standard techniques.

It will be understood that variations on the above procedures are usefulin the present invention. For example, it may be desired to transform ahost cell with DNA encoding either the light chain or the heavy chain(but not both) of an antibody. Recombinant DNA technology may also beused to remove some or all of the DNA encoding either or both of thelight and heavy chains that is not necessary for binding, e.g., theconstant region may be modified by, for example, deleting specific aminoacids. The molecules expressed from such truncated DNA molecules areuseful in the methods described herein. In addition, bifunctionalantibodies can be produced in which one heavy and one light chain bindto a toxin, and the other heavy and light chain are specific for anantigen other than the toxin, or another epitope of the toxin.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184, 187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al. (1988Science, 240:1041-1043); Liu et al. (1987) PNAS, 84:3439-3443; Liu etal., 1987, J. Immunol., 139:3521-3526; Sun et al. (1987) PNAS84:214-218; Nishimura et al., 1987, Canc. Res., 47:999-1005; Wood et al.(1985) Nature, 314:446-449; and Shaw et al., 1988, J. Natl. CancerInst., 80:1553-1559). Chimeric antibodies can also be created byrecombinant DNA techniques where DNA encoding murine V regions can beligated to DNA encoding the human constant regions.

An antibody or an immunoglobulin chain can be humanized by methods knownin the art. For example, once murine antibodies are obtained, variableregions can be sequenced. The location of the CDRs and frameworkresidues can be determined (see, Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol., 196:901-917). The light and heavy chainvariable regions can, optionally, be ligated to corresponding constantregions. Indeed, it is understood that any of the antibodies describedherein, including fully human antibodies, can be altered (e.g., bymutation, substitution) to contain a substitute constant region, e.g.,Fc region, or portion(s) thereof to achieve, for example, a desiredantibody structure, function (e.g., effector function), subtype,allotype, subclass, or the like. Anti-toxin antibodies can be sequencedusing art-recognized techniques. CDR-grafted antibody molecules orimmunoglobulins can be produced by CDR-grafting or CDR substitution,wherein one, two, or all CDRs of an immunoglobulin chain can bereplaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature,321:552-525; Verhoeyan et al., 1988, Science, 239:1534; Beidler et al.,1988, J. Immunol., 141:4053-4060; and Winter, U.S. Pat. No. 5,225,539.

Winter describes a CDR-grafting method that may be used to prepare theantibodies of the present invention (UK Patent Application GB 2188638A,filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents ofwhich is expressly incorporated by reference. For example, all of theCDRs of a particular antibody may be replaced with at least a portion ofa human CDR (e.g., a CDR from clone 3D8, as shown in Tables 1 and 2,and/or clone 124-152, as shown in Tables 3 and 4, above) or only some ofthe CDRs may be replaced. It is only necessary to replace the number ofCDRs required for binding of the antibody to a predetermined antigen(e.g., an exotoxin of C. difficile).

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L., 1985,Science, 229:1202-1207, by Oi et al., 1986, BioTechniques, 4:214, and byQueen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and U.S. Pat. No.5,693,762. Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulin Fvvariable regions from at least one of a heavy or light chain. Sources ofsuch nucleic acid are well known to those skilled in the art and, forexample, may be obtained from a hybridoma producing an antibody againsta predetermined target, as described above. The recombinant DNA encodingthe humanized antibody, or fragment thereof, can then be cloned into anappropriate expression vector. Other techniques for humanizingantibodies are described in Padlan et al. EP 519596 A1, published onDec. 23, 1992.

Also within the scope of the invention are antibodies in which specificamino acids have been substituted, deleted, or added. In particular,preferred antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, aselected, small number of acceptor framework residues of theimmunoglobulin chain can be replaced by the corresponding donor aminoacids. Preferred locations of the substitutions include amino acidresidues adjacent to the CDR, or which are capable of interacting with aCDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting aminoacids from the donor are described in U.S. Pat. No. 5,585,089 (e.g.,columns 12-16), the contents of which are hereby incorporated byreference. The acceptor framework can be a mature human antibodyframework sequence or a consensus sequence.

A “consensus sequence” is a sequence formed from the most frequentlyoccurring amino acids (or nucleotides) in a family of related sequences(See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft,Weinheim, Germany 1987). In a family of proteins, each position in theconsensus sequence is occupied by the amino acid occurring mostfrequently at that position in the family. If two amino acids occurequally frequently, either can be included in the consensus sequence. A“consensus framework” of an immunoglobulin refers to a framework regionin the consensus immunoglobulin sequence.

An anti-toxin antibody, or antigen-binding portion thereof, can bederivatized or linked to another functional molecule (e.g., anotherpeptide or protein). For example, an antibody can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that can mediate association with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized protein is produced by crosslinking two or moreproteins (of the same type or of different types). Suitable crosslinkersinclude those that are heterobifunctional, having two distinct reactivegroups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

Useful detectable agents with which a protein can be derivatized (orlabeled) include fluorescent compounds, various enzymes, prostheticgroups, luminescent materials, bioluminescent materials, and radioactivematerials. Exemplary fluorescent detectable agents include fluorescein,fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein orantibody can also be derivatized with detectable enzymes, such asalkaline phosphatase, horseradish peroxidase, β-galactosidase,acetylcholinesterase, glucose oxidase and the like. When a protein isderivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. A protein can also be derivatized with a prosthetic group(e.g., streptavidin/biotin and avidin/biotin). For example, an antibodycan be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding.

Labeled proteins and antibodies can be used, for example, diagnosticallyand/or experimentally in a number of contexts, including (i) to isolatea predetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; and (ii) to detect apredetermined antigen (e.g., a toxin, e.g., in a cellular lysate or apatient sample) in order to monitor protein levels in tissue as part ofa clinical testing procedure, e.g., to determine the efficacy of a giventreatment regimen.

An anti-toxin antibody or antigen-binding fragment thereof may beconjugated to another molecular entity, such as a label.

3. Screening Methods

Anti-toxin antibodies can be characterized for binding to the toxin by avariety of known techniques. Antibodies are typically characterized byELISA first. Briefly, microtiter plates can be coated with the toxin ortoxoid antigen in PBS, and then blocked with irrelevant proteins such asbovine serum albumin (BSA) diluted in PBS. Dilutions of plasma fromtoxin-immunized mice are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween 20 and then incubatedwith a goat-anti-human IgG Fc-specific polyclonal reagent conjugated toalkaline phosphatase for 1 hour at 37° C. After washing, the plates aredeveloped with ABTS substrate, and analyzed at OD of 405. Preferably,mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the toxin. Hybridomas that produce antibodies that bind,preferably with high affinity, to the toxin can than be subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify the anti-toxin antibodies, selected hybridomas can be grown inroller bottles, two-liter spinner-flasks or other culture systems.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.) topurify the protein. After buffer exchange to PBS, the concentration canbe determined by spectrophotometric methods.

To determine if the selected monoclonal antibodies bind to uniqueepitopes, each antibody can be biotinylated using commercially availablereagents (Pierce, Rockford, Ill.). Biotinylated MAb binding can bedetected with a streptavidin labeled probe. Anti-toxin antibodies can befurther tested for reactivity with the toxin by Western blotting.

Other assays to measure activity of the anti-toxin antibodies includeneutralization assays. In vitro neutralization assays can measure theability of an antibody to inhibit a cytopathic effect on cells inculture (see Example 3, below). In vivo assays to measure toxinneutralization are described in Examples 5, 6, and 7, below.

4. Pharmaceutical Compositions and Kits

In another aspect, the present invention provides compositions, e.g.,pharmaceutically acceptable compositions, which include an antibodymolecule described herein or antigen binding portion thereof, formulatedtogether with a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carriers” include any and all solvents,dispersion media, isotonic and absorption delaying agents, and the likethat are physiologically compatible. The carriers can be suitable forintravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, orepidermal administration (e.g., by injection or infusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Useful compositions are in the form of injectable or infusiblesolutions. A useful mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). For example,the antibody or antigen binding portion thereof can be administered byintravenous infusion or injection. In another embodiment, the antibodyor antigen binding portion thereof is administered by intramuscular orsubcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, theuseful methods of preparation are vacuum drying and freeze-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody portions described herein can beadministered by a variety of methods known in the art, and for manytherapeutic applications. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results.

In certain embodiments, an antibody, or antibody portion thereof may beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. Therapeutic compositions can be administered with medicaldevices known in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-60 mg/kg, e.g., 0.5-25 mg/kg, 1-2 mg/kg, or 0.75-10mg/kg. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Also within the scope of the invention are kits including an anti-toxinantibody or antigen binding portion thereof. The kits can include one ormore other elements including: instructions for use; other reagents,e.g., a label, a therapeutic agent, or an agent useful for chelating, orotherwise coupling, an antibody to a label or therapeutic agent, orother materials for preparing the antibody for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject.

Various combinations of antibodies can be packaged together. Forexample, a kit can include antibodies that bind to toxin A (e.g.,antibodies that include the variable heavy and light chain regions of3D8) and antibodies that bind to toxin B (e.g., human monoclonalanti-toxin B antibodies, e.g., 124-152, 2A11, and/or 1 G10, orpolyclonal antisera reactive with toxin B). The antibodies can be mixedtogether, or packaged separately within the kit.

Instructions for use can include instructions for therapeuticapplication including suggested dosages and/or modes of administration,e.g., in a patient with a symptom of CDAD. Other instructions caninclude instructions on coupling of the antibody to a chelator, a labelor a therapeutic agent, or for purification of a conjugated antibody,e.g., from unreacted conjugation components.

The kit can include a detectable label, a therapeutic agent, and/or areagent useful for chelating or otherwise coupling a label ortherapeutic agent to the antibody. Coupling agents include agents suchas N-hydroxysuccinimide (NHS). In such cases the kit can include one ormore of a reaction vessel to carry out the reaction or a separationdevice, e.g., a chromatographic column, for use in separating thefinished product from starting materials or reaction intermediates.

The kit can further contain at least one additional reagent, such as adiagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agentas described herein, and/or one or more additional anti-toxin or anti-C.difficile antibodies (or portions thereof), formulated as appropriate,in one or more separate pharmaceutical preparations.

Other kits can include optimized nucleic acids encoding anti-toxinantibodies, and instructions for expression of the nucleic acids.

5. Therapeutic Methods and Compositions

The new proteins and antibodies have in vitro and in vivo therapeutic,prophylactic, and diagnostic utilities. For example, these antibodiescan be administered to cells in culture, e.g., in vitro or ex vivo, orto a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/ordiagnose C. difficile and disease associated with C. difficile.

As used herein, the term “subject” is intended to include human andnon-human animals. The term “non-human animals” includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,chickens, mice, dogs, cats, pigs, cows, and horses.

The proteins and antibodies can be used on cells in culture, e.g., invitro or ex vivo. For example, cells can be cultured in vitro in culturemedium and the contacting step can be effected by adding the anti-toxinantibody or fragment thereof, to the culture medium. The methods can beperformed on virions or cells present in a subject, as part of an invivo (e.g., therapeutic or prophylactic) protocol. For in vivoembodiments, the contacting step is effected in a subject and includesadministering an anti-toxin antibody or portion thereof to the subjectunder conditions effective to permit binding of the antibody, orportion, to any toxin expressed by bacteria in the subject, e.g., in thegut.

Methods of administering antibody molecules are described herein.Suitable dosages of the molecules used will depend on the age and weightof the subject and the particular drug used. The antibody molecules canbe used as competitive agents for ligand binding to inhibit or reduce anundesirable interaction, e.g., to inhibit binding of toxins to thegastrointestinal epithelium.

The anti-toxin antibodies (or antigen binding portions thereof) can beadministered in combination with other anti-C. difficile antibodies(e.g., other monoclonal antibodies, polyclonal gamma-globulin).Combinations of antibodies that can be used include an anti-toxin Aantibody or antigen binding portion thereof and an anti-toxin B antibodyor antigen binding portion thereof. The anti-toxin A antibody can be3D8, an antibody that includes the variable regions of 3D8, or anantibody with variable regions at least 90% identical to the variableregions of 3D8. The anti-toxin B antibody can be 124-152, 2A11, 1 G10,or an antibody with variable regions at least 90% identical to thevariable regions of the foregoing, e.g., 124-152. Combinations ofanti-toxin A (e.g., 3D8) and anti-toxin B antibodies (e.g., 124-152) canprovide potent inhibition of CDAD.

It is understood that any of the agents of the invention, for example,anti-toxin A or anti-toxin B antibodies, or fragments thereof, can becombined, for example in different ratios or amounts, for improvedtherapeutic effect. Indeed, the agents of the invention can beformulated as a mixture, or chemically or genetically linked using artrecognized techniques thereby resulting in covalently linked antibodies(or covalently linked antibody fragments), having both anti-toxin A andanti-toxin B binding properties. The combined formulation may be guidedby a determination of one or more parameters such as the affinity,avidity, or biological efficacy of the agent alone or in combinationwith another agent. The agents of the invention can also be administeredin combination with other agents that enhance access, half-life, orstability of the therapeutic agent in targeting, clearing, and/orsequestering C. difficile or an antigen thereof.

Such combination therapies are preferably additive and even synergisticin their therapeutic activity, e.g., in the inhibition, prevention(e.g., of relapse), and/or treatment of C. difficile-related diseases ordisorders (see, e.g., Example 16 which shows the efficacy of single andcombined antibody therapies). Administering such combination therapiescan decrease the dosage of the therapeutic agent (e.g., antibody orantibody fragment mixture, or cross-linked or genetically fusedbispecific antibody or antibody fragment) needed to achieve the desiredeffect.

Immunogenic compositions that contain an immunogenically effectiveamount of a toxin, or fragments thereof, are described herein, and canbe used in generating anti-toxin antibodies. Immunogenic epitopes in atoxin sequence can be identified according to methods known in the art,and proteins, or fragments containing those epitopes can be delivered byvarious means, in a vaccine composition. Suitable compositions caninclude, for example, lipopeptides (e.g., Vitiello et al., J. Clin.Invest. 95:341 (1995)), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridgeet al., Molec. Immunol. 28:287-94 (1991); Alonso et al., Vaccine12:299-306 (1994); Jones et al., Vaccine 13:675-81 (1995)), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-75 (1990); Hu et al., Clin. Exp.Immunol. 113:235-43 (1998)), and multiple antigen peptide systems (MAPs)(see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-13 (1988); Tam,J. Immunol. Methods 196:17-32 (1996)).

Useful carriers that can be used with immunogenic compositions of theinvention are well known, and include, for example, thyroglobulin,albumins such as human serum albumin, tetanus toxoid, polyamino acidssuch as poly L-lysine, poly L-glutamic acid, influenza, hepatitis Bvirus core protein, and the like. The compositions can contain aphysiologically tolerable (i.e., acceptable) diluent such as water, orsaline, typically phosphate buffered saline. The compositions andvaccines also typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are examples of materials well known in the art. Additionally, CTLresponses can be primed by conjugating toxins (or fragments, inactivederivatives or analogs thereof) to lipids, such astripalmitoyl-S-glycerylcysteinyl-seryl-serine (P₃CSS).

The anti-toxin antibodies can be administered in combination with otheragents, such as compositions to treat CDAD. For example, therapeuticsthat can be administered in combination with anti-toxin antibodiesinclude antibiotics used to treat CDAD, such as vancomycin,metronidazole, or bacitracin. The antibodies can be used in combinationwith probiotic agents such as Saccharomyces boulardii. The antibodiescan also be administered in combinations with a C. difficile vaccine,e.g., a toxoid vaccine.

6. Other Methods

An anti-toxin antibody (e.g., monoclonal antibody) can be used toisolate toxins by standard techniques, such as affinity chromatographyor immunoprecipitation. Moreover, an anti-toxin antibody can be used todetect the toxin (e.g., in a stool sample), e.g., to screen samples forthe presence of C. difficile. Anti-toxin antibodies can be useddiagnostically to monitor levels of the toxin in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen.

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques in polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Generation of Anti-Toxin a Monoclonal Antibodies

C. difficile toxin A was obtained either from Techlab, Inc. (Blacksburg,Va.), or by recombinant production. The toxin was purified andinactivated prior to immunization. Inactivation was performed bytreatment with reactive UDP-dialdehyde, which results in alkylation ofcatalytic residues while preserving native toxin structure. For thedetailed protocol, see Genth et al., Inf and Immun. 68(3):1094-1101,2000. Briefly, purified toxin A was incubated with UDP-2′,3′-dialdehyde(0.1-1.0 mM) in buffer for 18 hours at 37° C., filtered through a 100kDa-cutoff filter to remove unreacted UDP-2′,3′-dialdehyde, and washedwith buffer. Inactivated toxin A (toxoid A) was used for immunization.

HCo7 transgenic mice, generated as described above in the sectionentitled “Generation of Human Monoclonal Antibodies in HuMAb Mice” andsupplied by Medarex, Milpitas, Calif., were immunized intraperitoneally6-12 times each with 10 μg of toxoid in RIBI adjuvant. In the HCo7transgenic mice, the endogenous mouse kappa light chain gene has beenhomozygously disrupted as described in Chen et al. (1993) EMBO J.12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of PCT Publication WO01/09187. The HCo7 transgenic mice carry a human kappa light chaintransgene, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851, and the HCo7 human heavy chain transgene asdescribed in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807. Serumwas collected from each mouse and tested for reactivity to toxin A byELISA and neutralization of cytotoxicity on IMR-90 cells. Mice thattested positive for toxin A-reactive and neutralizing antiserum wereinjected with 5-10 μg toxoid A through the tail vein. Mice weresacrificed and spleens were isolated for fusion to hybridomasapproximately 3 days after tail vein injection was performed.

Clonal hybridomas were generated and screened by ELISA. Percentages ofkappa/gamma light chain positive, antigen-specific, and neutralizingclones identified by screening clones generated from four separatehybridoma fusions are listed in Table 5.

TABLE 5 % kappa/gamma Fusion positive % antigen specific % neutralizing1 5.7 (94/1632)  3.4 (56/1632)  0.7 (12/1632) 2 0.2 (1/384)   0 (0/384) 0 (0/384) 3 1.8 (14/768) 0.39 (3/768)  4 4.4 (43/960) 1.7 (17/960)

Three hybridoma clones were selected for further analysis: 3D8, 1B11,and 33.3H2. CDNAs from each clone were amplified by RT-PCR from mRNA,cloned, and sequenced. One heavy chain V region consensus sequence wasfound for each clone. All three clones utilized a VH region derived fromthe same germline V region gene (VH 3-33), but utilized different Jsequences. The amino acid sequences of the VH and VL regions from eachclone are shown in FIG. 1 (SEQ ID NOs: 1-6). The complementaritydetermining regions (CDRs) are overlined in the Figure.

Sequence analysis of the kappa V (VK light chain) genes revealed thatHuMAb 1B11 and 33.3H2 each express one consensus kappa chain V sequence.The 1B11 hybridoma expressed a Vκ light chain derived from the Vκ L6germline gene, whereas the 33.3H2 hybridoma expresses a Vκ light chainderived from the Vκ L15 germline gene. Upon analysis of the Vκ clonesfrom HuMAb 3D8, 6 (I-VI) light chains were expressed at the mRNA level(FIG. 1). To determine which of the light chains were expressed at theprotein level, mass spectroscopy and N-terminal sequencing of thepurified 3D8 antibody were performed. When light chains were isolatedfrom cellular protein and analyzed by mass spectroscopy, a single lightchain was seen with a mass of 23,569 Daltons. This corresponded to thelight chain with the group I amino acid sequence depicted in FIG. 1,which is derived from the Vκ L19 germline gene. N-terminal sequencing ofthe light chain confirmed this result. FIGS. 2A, 3A, and 4A depict thenucleotide and the amino acid sequences of the Vκ of each 3D8 (group I;SEQ ID NOs: 4, and 30-34), 1B11 (SEQ ID NO: 5), and 33.3H2 (SEQ ID NO:6)respectively. The CDRs are overlined and the germline Vκ and Jκ areshown.

Thus, the 3D8 antibody comprises a heavy chain variable region that isthe product of or derived from a human VH 3-33 gene and a light chainvariable region that is the product of or derived from a human Vκ L19gene. The 1B11 antibody comprises a heavy chain variable region that isthe product of or derived from a human VH 3-33 gene and a light chainvariable region that this the product of or derived from a human Vκ L6gene. The 33.3H2 antibody comprises a heavy chain variable region thatis the product of or derived from a human VH 3-33 gene and a light chainvariable region that this the product of or derived from a human Vκ L15gene.

The antibodies 3D8 and 1B11 express human IgG1 constant regions, andantibody 33.3H2 expresses human IgG3 constant regions. The antibodiesdescribed in Examples 2-7 were isolated from these hybridomas, and thusexpress the variable sequences shown in FIG. 1 along with human constantregions. DNA encoding the antigen binding portion of each clone wascloned into a vector to be expressed as a human antibody foradministration to humans.

Example 2. Binding Activity of Anti-Toxin a Antibodies

Binding of each antibody to toxin A was determined by ELISA usingstandard techniques. The results of this assay are depicted in FIG. 5.Antibodies produced by 3D8, 1B11, and 33.3H2 were compared to a fourthhuman monoclonal antibody with toxin A binding activity, 8E6. FIG. 5shows that the antibodies bind toxin A with comparable affinities.

The affinity of the 3D8 and 1B11 antibodies for toxin A was alsomeasured with Biacore® instrument, which detects biomolecular bindinginteractions with surface plasmon resonance technology. Each antibodywas added to protein A-coated sensor chips, and toxin A was allowed toflow over the chip to measure binding. 3D8 had a K_(D) of 14.6×10⁻¹⁰M.1B11 had a K_(D) of 7.38×10⁻¹⁰M. Thus, the antibodies bind with highaffinity to toxin A. These binding constants indicate that theantibodies have affinities suitable for use in human therapy.

Example 3. Toxin Neutralization by Anti-Toxin a Antibodies

Antibodies expressed by 1B11, 3D8, and 33.3H2 hybridomas were tested fortoxin A neutralization activity in vitro. Cells were incubated in thepresence of varying concentrations of toxin A, which causes cells toround up and lose adherence to cell culture dishes. Cytopathic effect(CPE) was determined by visual inspection of cells. A CPE score from 0-4was determined, based on the results of the visual inspection (4=100%cytotoxicity, 0=0% toxicity). The results of these assays are depictedin FIGS. 6A and 6B. Neutralization of toxicity against a human lungfibroblast cell line, IMR-90, and a human gut epithelial cell line,T-84, was determined. FIG. 6A shows that all of the antibodies hadneutralizing capacity towards IMR-90 cells. The relative neutralizingactivity of toxin A cytotoxicity on IMR-90 cells was 1B11>3H2>3D8.Interestingly, the relative neutralizing activity was 3D8≥1B11>3H2against T-84 cells, which are human colonic epithelial cells (FIG. 6A).T-84 cells are believed to be more sensitive to toxin A than other celltypes. T-84 cells may provide a more relevant target cell to determinetoxin A cytotoxicity.

Example 4. Epitope Mapping of Anti-Toxin A Antibodies

The epitope of toxin A bound by each monoclonal antibody was determinedby western blotting. Recombinant E. coli clones were constructed whichexpress four fragments of toxin A representing the enzymatic domain(i.e., amino acids 1-659 of toxin A), the receptor binding domain (i.e.,amino acids 1853-2710 of toxin A), and the two regions in between (i.e.,amino acids 660-1255 and 1256-1852 of toxin A). The appropriate segmentsof the toxin A gene were PCR-amplified from genomic DNA prepared from C.difficile strain ATCC 43255. The fragments were cloned using a pETvector and transformed into BL21 DE3 cells for expression. The vectorprovides inducible expression and affinity domains for purification(i.e., a His-tag) and detection (i.e., a V5 epitope tag). Expression wasinduced with IPTG and fragments were purified by affinitychromatography. Binding to four different fragments of toxin A wasmeasured: fragment 1 corresponded to amino acids 1-659; fragment 2corresponded to amino acids 660-1255; fragment 3 corresponded to aminoacids 1256-1852; and fragment 4 corresponded to amino acids 1853-2710(FIG. 7). 1B11 reacted with fragments 1 and 2. 33.3H2 reacted withfragment 2. 3D8 and another human monoclonal antibody, 6B4, reacted withfragment 4 (the receptor binding domain). A polyclonal antiserum fromrabbits immunized with toxoid A reacted with all four fragments.

The 1B11 and 33.3H2 epitopes were mapped in further detail. To map the1B11 epitope, subfragments of fragment 1 (amino acids 1-659)corresponding to amino acids 1-540, 1-415, 1-290, and 1-165, weregenerated (FIG. 8A). 1B11 bound to fragment 1 and to the fragmentcontaining amino acids 1-540. 1B11 did not bind to the othersubfragments. Therefore, the epitope bound by 1B11 maps between aminoacids 415-540 of toxin A.

To map the 33.3H2 epitope, subfragments of fragment 2 (amino acids660-1255) corresponding to amino acids 660-1146, 660-1033, 660-920, and660-807, were generated (FIG. 8B). 33.3H2 bound to the fragmentscorresponding to amino acids 660-1255, 660-1146, and 660-1033. 33.3H2did not bind to the other subfragments. Therefore, the epitope bound by33.3H2 maps between amino acids 920-1033 of toxin A.

Example 5. Protection of Mice from Lethal Toxin a Challenge byAdministration of Anti-Toxin A Antibodies

Each antibody was tested for the ability to protect mice from challengewith a lethal dose of toxin A. Swiss Webster female mice, each weighing10-20 grams, were injected intraperitoneally with up to 250 μg of 3D8,1B11, or 33.3H2, or a control antibody (anti-respiratory syncytial virusantibody, Medlmmune) prior to challenge with toxin A. Approximately 24hours after injection, mice were challenged with a dose of toxin Agreater than 10 times the lethal dose (LD₅₀), typically 100 ng. Animalswere observed for signs of toxicity for the next 7 days. The results ofthese experiments are summarized in FIG. 9. The data is expressed aspercentage survival. Numbers in parenthesis refer to antibody dose, if adose other than 250 μg was given. FIG. 9 shows that each of theantibodies was able to protect mice from lethal toxin A challenge tosome extent. The percentage of mice surviving when treated with 3D8ranged from 10-100 percent. The percentage of mice surviving whentreated with 33.3H2 ranged from 20-100 percent. The percentage of micesurviving when treated with 1B11 ranged from 0-60 percent. The relativeability of these monoclonals to protect mice was 3H2≥3D8>1B11.

Example 6. Neutralization of Toxin A Enterotoxicity in Ligated MouseIntestinal Loops with Anti-Toxin A Antibodies

3D8 and 33.3H2 antibodies were tested for neutralization of toxin Aenterotoxicity in a mouse ileal loop model. This model measures toxinA-induced fluid accumulation in mouse intestine. To perform theseexperiments, each mouse was starved for 16 hours, anesthetized, and theileum next to the cecum was exposed. A loop of 3 to 5 centimeters wasdoubly ligated at each end and injected with 10 μg of toxin A. The ilealloop was returned to the abdominal cavity, the wound was closed, and theanimal was allowed to recover. Four hours after surgery, the animal waseuthanized and the loop was removed from the animal. The length of eachsegment was remeasured, and the intraluminal fluid was extracted. Thevolume of the fluid and the volume-to-length (V:L) ratio in millilitersper centimeter was calculated for each loop. Test mice were injectedwith antibody parenterally 1-2 days before surgery. The results of theseexperiments are depicted in FIG. 10. Injection with toxin A increasedthe weight to length ratio of intestinal fluid by 50%. Both 3D8 and33.3H2 prevented this increase in fluid accumulation. Mice administeredeither antibody had a weight to length ratio comparable to mice that didnot receive any toxin A injection. Therefore, 3D8 and 33.3H2 protectfrom intestinal fluid accumulation in vivo.

These results indicate that the anti-toxin A monoclonal antibodiesprotect from toxin A-mediated enterotoxicity in vivo. The mouse ligatedloop data shows that these monoclonal antibodies can protect frommucosal damage when administered systemically.

Example 7. Protection of Hamsters from C. difficile Relapse withAnti-Toxin A Antibodies

3D8 was tested in a hamster relapse model. Hamsters are sensitive to thetoxic effects of C. difficile toxins, and typically die within 2-3 daysof receiving a single dose of clindamycin in the presence of C.difficile. To test the efficacy of 3D8 in hamsters, a relapse model wasused. In this model, hamsters were given a dose of clindamycin and adose of C. difficile B1 spores one day later. One set of controlhamsters received no additional antibiotic or antibody. A second set ofcontrol hamsters were treated with 10 mg/kg/day vancomycin. Vancomycinis an antibiotic used in the treatment of C. difficile disease. As shownin FIG. 11A, a test set of hamsters received 10 mg/kg/day vancomycin and2 mg/kg/day of a rabbit polyclonal antiserum raised against toxin A eachday for seven days after C. difficile exposure, as indicated by thearrows in the figure. A second test set of hamsters received 10mg/kg/day vancomycin and 50 mg/kg/day 3D8 at the same time intervals.Hamster survival was plotted versus time and is shown in FIG. 11B.

FIG. 11B shows that all of the hamsters that received only clindamycinand C. difficile (diamonds) died within two days of challenge with thebacteria. Twelve percent (2/17) of hamsters treated with vancomycin(squares) survived challenge with bacteria; eighty-eight percent (15/17)died within eight days. Forty-one percent (7/17) of hamsters treatedwith vancomycin and 3D8 (crosses) survived challenge; fifty-nine (10/17)percent died within seven days. Sixty-four percent (7/11) of hamsterstreated with vancomycin and polyclonal rabbit serum (triangles) survivedthe challenge with bacteria; thirty-six percent (4/11) died within ninedays. These data are also depicted in FIG. 12 as the percentage of totalsurvivors in each treatment group. As shown in the figure, thepercentage of survivors was highest (sixty-four percent) in the groupreceiving vancomycin and polyclonal rabbit serum. The group receiving3D8 and vancomycin had the second highest rate of survival (forty-onepercent). Only twelve percent of vancomycin-treated hamsters survived.Those with no treatment all died. These data show that polyclonal andmonoclonal anti-toxin antibodies protect from relapse of C. difficiledisease in vivo when administered after infection.

Example 8. Production of Anti-Toxin A Antibodies for Administration inHumans

Nucleic acid sequences encoding the variable heavy chain and lightchains of the 3D8 antibody were cloned into a pIE-UgammalF vector usingstandard recombinant DNA methodology. The vector was amplified in E.coli, purified, and transfected into CHO-dg44 cells. Transfected cellswere plated at 4×10⁵ cells per well in a 96-well dish and selected forvector transfection with G418. One clone, designated 1D3, was originallyselected by G418 resistance, then assayed along with other transfectomasfor production of IgG. 1D3 had a higher level of IgG production relativeto other transfectants during several rounds of expansion. Theexpression of the 3D8 antibody was amplified by growth in the presenceof increasing concentrations of methotrexate. A culture capable ofgrowth in 175 nM methotrexate was chosen for cloning single cells forfurther development. Plating the culture in 96 well plates at lowdensity allowed generation of cultures arising from a single cell orclones. The cultures were screened for production of human IgG, and thecell that produced the highest level of IgG was selected for furtheruse. The methotrexate-amplified clone was expanded to produce a cellbank including multiple frozen vials of cells.

To prepare antibodies from transfected cells, cells from a cloneisolated in the previous steps are cultured and expanded as inoculum fora bioreactor. The bioreactor typically holds a 500 liter volume ofculture medium. The cells are cultured in the bioreactor until cellviability drops, which indicates a maximal antibody concentration hasbeen produced in the culture. The cells are removed by filtration. Thefiltrate is applied to a protein A column. Antibodies bind to thecolumn, and are eluted with a low pH wash. Next, the antibodies areapplied to a Q-Sepharose column to remove residual contaminants, such asCHO cell proteins, DNA, and other contaminants (e.g., viralcontaminants, if present). Antibodies are eluted from the Q-Sepharosecolumn, nano-filtered, concentrated, and washed in a buffer such as PBS.The preparation is then aseptically aliquoted into vials foradministration.

Example 9. Preparation and Characterization of Polyclonal Anti-Toxin BAntibodies

Two Nubian goats (#330 and #331) were injected intramuscularly with 50μg UDP dialdehyde-inactivated toxin B (Techlab) and complete Freund'sadjuvant. Booster doses of 25 μg toxoid B with Freund's incompleteadjuvant were given intramuscularly at two-week intervals. Test bleedswere obtained after 4 immunizations. ELISA reactivity and neutralizationof cytotoxicity against both toxin A and toxin B were assayed to measurethe specificity and cross reactivity of the sera.

Both animals responded well to toxin B and to a lesser extent to toxin Aas measured by ELISA. Sera from goat #331 had less toxin Across-reactivity and was chosen for the majority of the subsequentexperiments. Neutralization of cytotoxicity to IMR-90 cells wasdetermined as described in Example 3. The results of cytotoxicityneutralization are depicted in FIG. 13, which shows that sera from bothanimals exhibited good toxin B neutralizing antibody titers and verylow, but detectable, toxin A neutralizing antibody titers. The abilityof the goat sera to protect mice from a lethal intraperitoneal challengewith toxin B (100 ng) was also confirmed (data not shown).

Example 10. Protection of Hamsters from C. difficile Relapse withAnti-Toxin a and Anti-Toxin B Antibodies

Groups of hamsters (n=20) were challenged with clindamycin and C.difficile, and then treated with vancomycin as described in the hamstermodel of relapse in Example 7. Antibodies (either 3D8, serum from goat#331, or 3D8 and serum from goat #331) were given twice daily aftervancomycin treatment (FIG. 14). Animals were monitored for survival(FIG. 15) or illness (FIG. 16). Antibody doses were 1 ml twice daily forserum from goat #331 and 3 mg for 3D8 given twice daily. Animalsreceiving vancomycin only (i.e., no antibody treatment) served as anegative controls. As observed previously, 3D8 and vancomycin treatmentalone demonstrated a partial protective effect, in which 10 out of 20animals were protected from lethality (FIG. 15). Fifty percent ofanimals in this group remained healthy (FIG. 16). Six out of 20 animalsreceiving vancomycin treatment alone were protected (FIG. 15). Thirtypercent remained healthy (FIG. 16). Partial protection (9/20 animalsprotected) was also observed when the goat serum was used alone (FIG.15). Forty percent remained healthy. Protection was increased to nearly100% when both goat serum and 3D8 were given together (18/20) anddisease onset was delayed (FIG. 15). Ninety percent of these animalsremained healthy (FIG. 16). Clearly, protection from illness followed apattern similar to protection from lethality. These data demonstratethat 3D8 can be fully protective in the hamster disease model when toxinB is also neutralized.

Example 11. Protection of Hamsters from C. difficile Relapse in HamstersImmunized with Toxin B

Hamsters were immunized intraperitoneally with 10 μg of theCOOH-terminal fragment of toxin B (corresponding to amino acids1777-2366 of toxin B) expressed in E. coli and using RIBI as adjuvant.Animals received 7 doses of toxin B antigen. Neutralizing antibodyresponses were observed in the animals that were tested. Groups ofimmunized hamsters were challenged with clindamycin and C. difficilethen treated with vancomycin as described in the hamster model ofrelapse in Example 7. Antibody (3D8, 3 mg/dose) was given twice dailyafter vancomycin treatment to 19 animals and compared to a negativecontrol group (n=20) that received no treatment (FIGS. 17 and 18). Sixanimals were challenged without vancomycin treatment to ensure thathamsters immunized with toxin B antigen were susceptible to C. difficileinfection. Animals were monitored for survival (FIG. 17) or illness(FIG. 18). FIG. 17 shows that immunized animals that were not given 3D8relapsed at a similar rate to that observed previously (65% relapse).Toxin B-immunized animals receiving 3D8 were more fully protected fromrelapse than observed previously (10% relapse, as compared toapproximately 50% relapse in animals not previously immunized with toxinB in other experiments).

FIG. 18 shows that some of the immunized animals receiving 3D8 becameill but recovered from their diarrhea. Thirty five percent of immunizedanimals receiving vancomycin alone remained healthy. In experiments inwhich toxin B reactive sera were not present in animals, virtually allanimals that had diarrhea later died. These data provide furtherevidence that 3D8 can be fully protective in the hamster disease modelwhen toxin B is also neutralized. Neutralization of toxin B in additionto toxin A was required for optimal protection from C. difficile diseasein this model.

Example 12. Protection of Hamsters from Primary C. Difficile ChallengeUsing 3D8 in Hamsters Treated with Goat Anti-Toxin B Sera

Prevention of relapse of C. difficile disease in the hamsters was easierto demonstrate than protection from direct challenge (i.e., challengewithout vancomycin administration). Experiments with rabbit serademonstrated only weak protection from direct challenge and 3D8 had nodetectable affect on direct challenge. Since 3D8 was more protective ina background of toxin B neutralizing antibodies, it was determinedwhether the combined administration of 3D8 and anti-toxin B antiseracould prevent disease due to direct challenge. Groups of 5 hamsters werechallenged after receiving once daily doses of 3D8 (3 mg), combined 3D8(3 mg) and goat #331 (1 ml) sera, or no antibodies for the 3 days priorto challenge as depicted in FIG. 19. The data in FIG. 20 shows thatanimals receiving no antibodies or either 3D8 or goat sera alone alldied with 48 hours of C. difficile challenge. Most animals (80%)receiving both 3D8 and goat sera survived and the affected animalssurvived for 10 days after challenge. FIG. 21 shows that animals treatedwith 3D8 and goat sera became ill but recovered. These data providefurther evidence that 3D8 can be fully protective in the hamster diseasemodel when toxin B is also neutralized. Neutralization of toxin B inaddition to toxin A was required for optimal protection from C.difficile disease in this model.

The successful protection of hamsters directly challenged with C.difficile offers several advantages to the screening of new toxin Bcandidates. Smaller numbers of animals can be used since 100% ofuntreated animals die. Antibodies, such as monoclonal antibodies (e.g.,human monoclonal antibodies) can be screened directly in hamstersbecause the procedure requires 100 mg or less of the test antibody.Other modes of testing, such as the relapse model, require the effort ofproducing gram quantities due to the low attack rate in the relapsemodel, which necessitates testing larger numbers of animals. Directchallenge experiments are also shorter in duration with a definitiveread out within 3-4 days of C. difficile challenge compared to 7-10 inthe relapse model. In addition, the elimination of vancomycin treatmentfrom the screening method reduces the number of times animals arehandled.

Example 13. Generation of Anti-Toxin B Monoclonal Antibodies

C. difficile toxin B was obtained either from Techlab, Inc. (Blacksburg,Va.), or by recombinant production. The toxin was purified andinactivated prior to immunization. Inactivation was performed bytreatment with reactive UDP-dialdehyde, which results in alkylation ofcatalytic residues while preserving native toxin structure. Briefly,purified toxin B was incubated with UDP-2′,3′-dialdehyde (0.1-1.0 mM) inbuffer for 18 hours at 37° C., filtered through a 100 kDa-cutoff filterto remove unreacted UDP-2′,3′-dialdehyde, and washed with buffer.Inactivated toxin B (toxoid B) or recombinant toxin B fragments wereused as immunogens. A toxin B receptor binding domain (amino acidresidues 1777-2366) was expressed in E. coli as a fusion proteincontaining an immunotag (hexahistadine) for affinity purification usingnickel chelate affinity chromatography (designated fragment 4; seeExample 11).

Hco12 transgenic mice, generated as described above in the sectionentitled “Generation of Human Monoclonal Antibodies in HuMAb Mice” andsupplied by Medarex, Milpitas, Calif., were immunized intraperitoneally6-12 times each with 10 μg of toxoid in RIBI adjuvant. In the Hco12transgenic mice, the endogenous mouse kappa light chain gene has beenhomozygously disrupted as described in Chen et al. (1993) EMBO J.12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of PCT Publication WO01/09187. The Hco12 transgenic mice carry a human kappa light chaintransgene, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851, and the Hco12 human heavy chain transgene asdescribed in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807. Serumwas collected from each mouse and tested for reactivity to toxin B byELISA and neutralization of cytotoxicity on IMR-90 cells. Mice thattested positive for toxin B-reactive and neutralizing antiserum wereinjected with 5-10 μg toxoid B or fragment 4 through the tail vein. Micewere sacrificed and spleens were isolated for fusion to hybridomasapproximately 3 days after tail vein injection was performed.

Clonal hybridomas were generated and screened by ELISA. Three hybridomaclones were selected for further analysis: 124-152; 2A11; and 1 G10. Inparticular, cDNAs from the 124-152 clone were amplified by RT-PCR frommRNA, cloned, and sequenced. The heavy chain V region was determined tobe derived from the germline sequence VH 5-51, the D region derived fromthe germline sequence 7-27, and the J sequence from the germline regionJH3b. The light chain (kappa) regions were determined to be derived fromA27 and the J region from JK1. The isotype of the 124-152 clone wasdetermined to be IgG1. The amino acid sequences of the VH and VL regionsof the 124-152 clone are shown in FIGS. 27-28. The complementaritydetermining regions (CDRs) are indicated in the Figures. The relatedgermline sequences of the VH and VL regions are shown in FIGS. 30-31.

The antibodies 124-152; 2A11; and 1 G10 were isolated from correspondinghybridomas and tested for their binding characteristics (infra). DNAencoding the 124-152 clone was cloned into a vector to be expressed as ahuman antibody for administration to humans.

Example 14. Binding Activity of Anti-Toxin B Antibodies

Binding of each antibody to toxin B was determined by Biacore usingstandard techniques. The results of this assay are depicted in Table 6.Antibodies produced by 124-152; 2A11; and 1 G10 were compared toappropriate controls.

In particular, the affinity of the 124-152; 2A11; and 1 G10 antibodiesfor toxin B was measured with Biacore® instrument, which detectsbiomolecular binding interactions with surface plasmon resonancetechnology. Each antibody was added to protein A-coated sensor chips,and toxin B was allowed to flow over the chip to measure binding.124-152 had a K_(D) of 1.64×10⁻¹⁰M; 2A11 had a K_(D) of 0.24×10⁻¹⁰M; and1 G10 had a K_(D) of 2.98×10⁻¹⁰M. Thus, the antibodies bind with highaffinity to toxin B. These binding constants indicate that theantibodies have affinities suitable for use in vivo application, forexample, human therapy.

TABLE 6 K_(D) × 10⁻¹⁰ k_(a) × 10⁵ k_(d) × 10⁻⁵ Sample ID (M) (1/Ms)(1/s) 2A11 0.24 21 5.07 124.152 1.64 34.5 56.4 51.1G10 2.98 1.31 3.89

Example 15. Toxin Neutralization by Anti-Toxin B Antibodies

Antibodies expressed by 124-152; 2A11; and 1 G10 hybridomas were testedfor toxin B neutralization activity in vitro. Cells were incubated inthe presence of varying concentrations of a monoclonal antibody specificto toxin B which would prevent cells from rounding up after exposure totoxin B. Cytopathic effect (CPE) was determined by visual inspection ofcells. A CPE score from 0-4 was determined, based on the results of thevisual inspection (4=100% cytotoxicity, 0=0% toxicity). The results ofthese assays are depicted in FIG. 27. Neutralization of toxicity againsta human lung fibroblast cell line, IMR-90. FIG. 27 shows that all of theantibodies had neutralizing capacity towards IMR-90 cells. The relativeneutralizing activity of toxin A cytotoxicity on IMR-90 cells was124-152>1 G10>2A11.

Example 16. Protection of Hamsters from Primary C. Difficile ChallengeUsing Anti-Toxin B Antibodies

Protection from direct challenge of an inoculum of C. difficile(clindamycin on day −1 and C. difficile spores on day O (1/100,000dilution) was performed over a period of 4 to 10 days in the presence orabsence of anti-toxin B antibodies. Groups of 5 hamsters were challengedafter receiving once daily doses of 3D8 (20 mg total over 4 days),combined 3D8 (Id.) and goat #331 (3 ml) sera, 3D8 in combination withanti-toxin B antibodies 124-152 (18 mg total over 4 days), 2A11 (20 mgtotal over 4 days), or 1 G10 (20 mg total over 4 days) or no antibodiesfor 3 days prior to challenge as depicted in FIG. 24. The data in FIG.24 shows that animals receiving no antibodies or either 3D8 or goat seraalone all died within 72 hours of C. difficile challenge whereas animalsreceiving 3D8 and an anti-toxin B antibody, and preferably incombination with 124-152, had a 40% survival rate (FIG. 24). A 10 daystudy similar to the foregoing (but using a more dilute C. difficileinoculum) was performed with increasing amounts of the anti-toxin Bantibody 124-152 (0.56 mg, 1.7 mg, or 5.0 mg given at days −3, −2, −1,and 0). Animals receiving both 3D8 and goat sera survived and mostanimals (60%-70%) survived for 10 days after challenge if given 3D8 incombination with 124-152. Even the lowest dosage of the anti-toxin Bantibody 124-152 (0.56 mg in combination with 3D8) was highly effective(70% survival; see FIG. 25). Results show that 124-152 and 3D8, alone,are less effective then when used in combination where a more thanadditive, indeed, synergistic therapeutic result is achieved (FIGS.24-26). These data provide further evidence that the anti-toxin Bantibody is highly effective, especially in combination with theanti-toxin A antibody 3D8. Neutralization of toxin B in addition totoxin A was determined to provide for protection from C. difficiledisease in this model.

Example 17. Epitope Mapping of Anti-Toxin B Antibodies

The epitope of toxin B bound by each monoclonal antibody was determinedby western blotting. Recombinant E. coli clones were constructed whichexpress fragments of toxin B representing different domains of toxin B.The appropriate segments of the toxin B gene were PCR-amplified from DNAprepared from an appropriate C. difficile strain. The fragments werecloned into an expression vector and expressed in E. coli. Humanmonoclonal antibody 152 was used to probe toxin B fragment in westernblots in order to map the binding epitope. Toxin B protein fragmentswere isolated from E. coli containing a portion of the toxin B genes andseparated using SDS-PAGE. After electrophoresis, the toxin B fragmentswere transferred to nitrocellulose and probed with monoclonal antibody152 followed by alkaline phosphatase conjugated goat anti human todetect MAb 152 binding. HuMab 152 was determined to bind to the —COOHfragment portion of toxin B between amino acids 1777 and 2366 (see, forexample, FIG. 32).

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. An isolated monoclonal antibody that binds toClostridium difficile (C. difficile) toxin A, or an antigen bindingportion thereof, wherein the antibody, or antigen binding portionthereof, comprises: (i) a heavy chain variable region CDR1 comprisingSEQ ID NO: 7; a heavy chain variable region CDR2 comprising SEQ ID NO:8; a heavy chain variable region CDR3 comprising SEQ ID NO: 9; a lightchain variable region CDR1 comprising SEQ ID NO: 16; a light chainvariable region CDR2 comprising SEQ ID NO: 17; and a light chainvariable region CDR3 comprising SEQ ID NO: 18; (ii) a heavy chainvariable region CDR1 comprising SEQ ID NO: 10; a heavy chain variableregion CDR2 comprising SEQ ID NO: 11; a heavy chain variable region CDR3comprising SEQ ID NO: 12; a light chain variable region CDR1 comprisingSEQ ID NO: 19; a light chain variable region CDR2 comprising SEQ ID NO:20; and a light chain variable region CDR3 comprising SEQ ID NO: 21; or(iii) a heavy chain variable region CDR1 comprising SEQ ID NO: 13; aheavy chain variable region CDR2 comprising SEQ ID NO: 14; a heavy chainvariable region CDR3 comprising SEQ ID NO: 15; a light chain variableregion CDR1 comprising SEQ ID NO: 22; a light chain variable region CDR2comprising SEQ ID NO: 23; and a light chain variable region CDR3comprising SEQ ID NO:
 24. 2. The isolated monoclonal antibody, or anantigen binding portion thereof, of claim 1, wherein the antibody, orantigen binding portion thereof, comprises a heavy chain variable regionCDR1 comprising SEQ ID NO: 7, a heavy chain variable region CDR2comprising SEQ ID NO: 8, a heavy chain variable region CDR3 comprisingSEQ ID NO: 9, a light chain variable region CDR1 comprising SEQ ID NO:16, a light chain variable region CDR2 comprising SEQ ID NO: 17, and alight chain variable region CDR3 comprising SEQ ID NO:
 18. 3. Theisolated monoclonal antibody, or an antigen binding portion thereof, ofclaim 1, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 10, aheavy chain variable region CDR2 comprising SEQ ID NO: 11, a heavy chainvariable region CDR3 comprising SEQ ID NO: 12, a light chain variableregion CDR1 comprising SEQ ID NO: 19, a light chain variable region CDR2comprising SEQ ID NO: 20, and a light chain variable region CDR3comprising SEQ ID NO:
 21. 4. The isolated monoclonal antibody, or anantigen binding portion thereof, of claim 1, wherein the antibody, orantigen binding portion thereof, comprises a heavy chain variable regionCDR1 comprising SEQ ID NO: 13, a heavy chain variable region CDR2comprising SEQ ID NO: 14, a heavy chain variable region CDR3 comprisingSEQ ID NO: 15, a light chain variable region CDR1 comprising SEQ ID NO:22, a light chain variable region CDR2 comprising SEQ ID NO: 23, and alight chain variable region CDR3 comprising SEQ ID NO:
 24. 5. Anisolated monoclonal antibody that binds to Clostridium difficile (C.difficile) toxin A, or an antigen binding portion thereof, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainvariable region having an amino acid sequence at least 95% identical tothe amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQID NO: 3 and/or a light chain variable region having an amino acidsequence at least 95% identical to the amino acid sequence set forth inSEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:
 6. 6. The isolated monoclonalantibody, or an antigen binding portion thereof, of claim 5, wherein theantibody, or antigen binding portion thereof, comprises a heavy chainvariable region having an amino acid sequence at least 95% identical tothe amino acid sequence set forth in SEQ ID NO:
 1. 7. The isolatedmonoclonal antibody, or an antigen binding portion thereof, of claim 5,wherein the antibody, or antigen binding portion thereof, comprises aheavy chain variable region having an amino acid sequence at least 95%identical to the amino acid sequence set forth in SEQ ID NO:
 2. 8. Theisolated monoclonal antibody, or an antigen binding portion thereof, ofclaim 5, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain variable region having an amino acid sequence atleast 95% identical to the amino acid sequence set forth in SEQ ID NO:3.
 9. The isolated monoclonal antibody, or an antigen binding portionthereof, of claim 5, wherein the antibody, or antigen binding portionthereof, comprises a light chain variable region having an amino acidsequence at least 95% identical to the amino acid sequence set forth inSEQ ID NO:
 4. 10. The isolated monoclonal antibody, or an antigenbinding portion thereof, of claim 5, wherein the antibody, or antigenbinding portion thereof, comprises a light chain variable region havingan amino acid sequence at least 95% identical to the amino acid sequenceset forth in SEQ ID NO:
 5. 11. The isolated monoclonal antibody, or anantigen binding portion thereof, of claim 5, wherein the antibody, orantigen binding portion thereof, comprises a light chain variable regionhaving an amino acid sequence at least 95% identical to the amino acidsequence set forth in SEQ ID NO:
 6. 12. The isolated monoclonalantibody, or an antigen binding portion thereof, of claim 5, wherein theantibody, or antigen binding portion thereof, comprises heavy and lightchain variable regions comprising amino acid sequences at least 95%identical to the amino acid sequences set forth in SEQ ID NOs: 1 and 4,respectively.
 13. The isolated monoclonal antibody, or an antigenbinding portion thereof, of claim 5, wherein the antibody, or antigenbinding portion thereof, comprises heavy and light chain variableregions comprising amino acid sequences at least 95% identical to theamino acid sequences set forth in SEQ ID NOs: 2 and 5, respectively. 14.The isolated monoclonal antibody, or an antigen binding portion thereof,of claim 5, wherein the antibody, or antigen binding portion thereof,comprises heavy and light chain variable regions comprising amino acidsequences at least 95% identical to the amino acid sequences set forthin SEQ ID NOs: 3 and 6, respectively.
 15. The isolated monoclonalantibody, or antigen binding portion thereof, of claim 1, wherein theantibody is a human antibody, a humanized antibody or a chimericantibody.
 16. The antigen binding portion of claim 1, wherein theantigen binding portion is a Fab, Fab′2, ScFv, Fd, Fv or dAb.
 17. Theisolated monoclonal antibody of claim 1, wherein the antibody is an IgG1or IgG3 isotype.
 18. The isolated monoclonal antibody, or antigenbinding portion thereof, of claim 1, wherein the K_(D) of the antibody,or antigen binding portion thereof, is less than 20×10⁻⁶ M.
 19. Theisolated monoclonal antibody, or antigen binding portion thereof, ofclaim 1, wherein the antibody, or antigen binding portion thereof,neutralizes toxin A in vitro or in vivo.